Transgenic maize event mon 87427 and the relative development scale

ABSTRACT

The invention provides transgenic maize event MON 87427 and plants, plant cells, seeds, plant parts, and commodity products derived from event MON 87427. The invention also provides nucleotides specific for transgenic maize event MON 87427 and plants, plant cells, seeds, plant parts, and commodity products comprising nucleotides specific for transgenic maize event MON 87427. The invention also provides methods related to transgenic maize event MON 87427 and to the Roundup® Hybridization System (RHS). The invention also provides a Relative Development Scale useful for monitoring and determining reproductive development in maize that reconciles developmental differences across various maize varieties. This is useful for determining the optimal timing of a treatment regimen in which tassel development stage is an important factor, including various methods in making hybrid seed.

CROSS REFERENCE TO RELATED APPLICATIONS

This application claims the benefit of U.S. Provisional Application No.61/263,526 filed Nov. 23, 2009, which is hereby incorporated byreference in its entirety herein, and U.S. Provisional Application No.61/263,530 filed Nov. 23, 2009, which is hereby incorporated byreference in its entirety herein.

INCORPORATION OF SEQUENCE LISTING

The sequence listing that is contained in the file named“56887-0001_seqlisting.txt”, which is 19.6 kilobytes (size as measuredin Microsoft Windows®) and was created on Nov. 12, 2010, is filedherewith by electronic submission and is hereby incorporated byreference in its entirety herein.

FIELD OF THE INVENTION

The invention relates to the fields of plant breeding, research, andagriculture. More specifically, it relates to transgenic maize event MON87427 and the nucleotide molecules, plants, plant parts, seeds, cells,agricultural products, and methods related to transgenic maize event MON87427. It also relates to predicting maize tassel development andutilizing this in the methods of plant breeding, research, andagriculture and the maize hybrid seed produced thereby.

BACKGROUND OF THE INVENTION

Crops having new, desirable traits are useful for plant breeding,research, and agricultural purposes. Such crops may be produced usingthe methods of biotechnology. However, the production and selection of acommercially suitable transgenic event may require extensive research,analysis, and characterization of a large number of individual planttransformation events in order to select an event having both thedesirable trait and the optimal phenotypic and agriculturalcharacteristics necessary to make it suitable for commercial andagricultural purposes. This process of event selection often requiresgreenhouse and field trials with many events over multiple years, inmultiple locations, and under a variety of conditions so that asignificant amount of agronomic, phenotypic, and molecular data may becollected. The resulting data and observations must then be analyzed byteams of scientists and agronomists with the goal of selecting acommercially suitable event. The invention provides such a commerciallysuitable event resulting in a new, desirable trait in maize.

Accurate determination of maize reproductive maturity is also useful forplant breeding, research, and agricultural purposes, such as in maizehybrid seed production. Tools commonly used in the art for predictingand estimating stages of maize growth and development include scalessuch as V-Stages, which are based on vegetative characteristics, andGrowing Degree Units, which are based on the number of growing degreedays. However, both of these tools produce estimates of tasseldevelopment stage that vary significantly across maize genotypes.Relying on these measurements may thus create a risk of missing theoptimally efficacious time for treatment regimens in which developmentstage is an important factor. The invention provides a RelativeDevelopment Scale based on tassel development reconciled acrossgenotypes, which is useful for monitoring and predicting tasseldevelopment in maize across various genotypes.

SUMMARY OF THE INVENTION

The invention provides a recombinant DNA molecule comprising a nucleicacid molecule comprising a nucleotide sequence selected from the groupconsisting of SEQ ID NO: 1-8. The invention also provides a recombinantDNA molecule formed by the junction of an inserted heterologous nucleicacid molecule and genomic DNA of a maize plant, plant cell, or seed. Theinvention also provides a recombinant DNA molecule derived fromtransgenic maize event MON 87427, a representative sample of seed havingbeen deposited with the American Type Culture Collection (ATCC®) underAccession No. PTA-7899. The invention also provides a recombinant DNAmolecule that is an amplicon diagnostic for the presence of DNA derivedfrom transgenic maize event MON 87427. The invention also provides arecombinant DNA molecule that is in a maize plant, plant cell, seed,progeny plant, plant part, or commodity product derived from transgenicmaize event MON 87427.

The invention also provides a DNA molecule comprising a nucleic acidmolecule having a nucleotide sequence of sufficient length of contiguousnucleotide sequence of SEQ ID NO: 10 to function as a DNA probe thathybridizes under stringent hybridization conditions with a DNA moleculecomprising a nucleotide sequence selected from the group consisting ofSEQ ID NO: 1-10 and does not hybridize under the stringent hybridizationconditions with a DNA molecule not comprising a nucleotide sequenceselected from the group consisting of SEQ ID NO: 1-10.

The invention also provides a pair of DNA molecules consisting of afirst DNA molecule and a second DNA molecule different from the firstDNA molecule, wherein the first and second DNA molecules each comprise anucleic acid molecule having a nucleotide sequence of sufficient lengthof contiguous nucleotides of SEQ ID NO: 10 to function as DNA primerswhen used together in an amplification reaction with DNA derived fromevent MON 87427 to produce an amplicon diagnostic for transgenic maizeevent MON 87427 DNA in a sample.

The invention also provides a method of detecting the presence of a DNAmolecule derived from MON 87427 in a sample by contacting a sample withthe DNA probe, subjecting the sample and the DNA probe to stringenthybridization conditions, and detecting hybridization of the DNA probeto a DNA molecule in the sample, wherein the hybridization of the DNAprobe to the DNA molecule indicates the presence of a DNA moleculederived from transgenic maize event MON 87427 in the sample.

The invention also provides a method of detecting the presence of a DNAmolecule derived from transgenic maize event MON 87427 in a sample bycontacting a sample with the pair of DNA molecules, performing anamplification reaction sufficient to produce a DNA amplicon comprising asequence selected from the group consisting of SEQ ID NO: 1-10, anddetecting the presence of the DNA amplicon in the reaction, wherein thepresence of the DNA amplicon in the reaction indicates the presence of aDNA molecule derived from MON 87427 in the sample.

The invention also provides a DNA detection kit comprising at least oneDNA molecule comprising a nucleotide sequence of sufficient length ofcontiguous nucleotide sequence of SEQ ID NO: 10 to function as a DNAprimer or probe specific for detecting the presence of DNA derived fromtransgenic maize event MON 87427, wherein detection of the DNA isdiagnostic for the presence of the transgenic maize event MON 87427 DNAin a sample.

The invention also provides a recombinant maize plant, seed, cell, orplant part thereof comprising a nucleic acid molecule having anucleotide sequence selected from the group consisting of SEQ ID NO:1-10. The invention also provides a recombinant maize plant, seed, cell,or plant part having tissue-selective tolerance to glyphosate herbicidetreatment. The invention also provides a recombinant maize plant, seed,cell, or plant part, the genome of which produces an amplicon comprisinga DNA molecule selected from the group consisting of SEQ ID NO: 1-10when tested in a DNA amplification method.

The invention also provides a maize plant or seed, wherein the maizeplant or seed is derived from transgenic maize event MON 87427. Theinvention also provides a maize plant or seed, wherein the maize plantor seed is a hybrid having at least one parent derived from transgenicmaize event MON 87427.

The invention also provides a nonliving plant material comprising arecombinant DNA molecule selected from the group consisting of SEQ IDNO: 1-10.

The invention also provides a microorganism comprising a nucleic acidmolecule having a nucleotide sequence selected from the group consistingof SEQ ID NO: 1-10. The invention also provides a microorganism that isa plant cell.

The invention also provides a commodity product produced from transgenicmaize event MON 87427 and comprising a nucleic acid molecule having anucleotide sequence selected from the group consisting of SEQ ID NO:1-10, wherein detection of a nucleotide sequence in a sample derivedfrom a commodity product is determinative that the commodity product wasproduced from transgenic maize event MON 87427. The invention alsoprovides a commodity product selected from the group consisting of wholeor processed seeds, animal feed, oil, meal, flour, flakes, bran,biomass, and fuel products. The invention also provides a method ofproducing a commodity product by obtaining a maize plant or part thereofcomprising transgenic maize event MON 87427 and producing a maizecommodity product from the maize plant or part thereof.

The invention also provides a method for controlling weeds in a field byplanting MON 87427 plants in a field and applying an effective dose ofglyphosate herbicide to control weeds in the field without injuring thetransgenic maize event MON 87427 plants.

The invention also provides a method for controlling weeds in a field,wherein the effective dose of glyphosate herbicide is from about 0.1pound to about 4 pounds per acre.

The invention also provides a method of producing a maize plant thattolerates application of glyphosate herbicide by sexually crossing atransgenic maize event MON 87427 plant comprising a nucleic acidmolecule comprising a nucleotide sequence selected from the groupconsisting of SEQ ID NO: 1-10 with a second maize plant, therebyproducing seed, collecting the seed produced from the cross, growing theseed to produce a plurality of progeny plants, treating the progenyplants with glyphosate, and selecting a progeny plant that is tolerantto glyphosate. The invention also provides a method of producing a maizeplant that tolerates application of glyphosate herbicide by selfing atransgenic maize event MON 87427 plant comprising a nucleic acidmolecule comprising a nucleotide sequence selected from the groupconsisting of SEQ ID NO: 1-10, thereby producing seed, collecting theseed produced from the selfing, growing the seed to produce a pluralityof progeny plants, treating the progeny plants with glyphosate, andselecting a progeny plant that is tolerant to glyphosate.

The invention also provides a method of producing hybrid maize seed byplanting transgenic maize event MON 87427 seed in an area, growing amaize plant from the seed, treating the plant with an effective dose ofglyphosate herbicide prior to pollen formation to make the plant malesterile without injuring the plant, fertilizing the plant with pollenfrom a second parent plant, and harvesting seed from the plant, whereinthe seed is hybrid maize seed produced by the cross of transgenic maizeevent MON 87427 plants with a second parent plant. The invention alsoprovides a method of producing hybrid maize seed, wherein the effectivedose of glyphosate herbicide is from about 0.1 pound to about 4 poundsper acre. The invention also provides a method of producing hybrid maizeseed, further including planting a second parent plant seed in the areaand growing a maize plant from the second parent plant. The inventionalso provides a method of producing hybrid maize seed, wherein thesecond parent plant is glyphosate tolerant.

The invention also provides a method for predicting the timing of maizetassel development by selecting a range on a Relative Development Scale,wherein the range indicates maturation to a desired tassel developmentstage. The invention also provides a method for predicting the timing ofmaize tassel development, wherein the desired tassel development stageis the optimal tassel development stage for reproductive crossing,tassel sterilization, detasseling, and/or administration of adevelopment modulating treatment to a maize plant. The invention alsoprovides a method for predicting the timing of maize tassel development,wherein the specific flower development stage used to construct theRelative Development Scale is at pollen shed for about 50 percent of apopulation of maize plants and wherein the range is about 0.62 and about0.75 on the Relative Development Scale. The invention also provides amethod for predicting the timing of maize tassel development, furtherincluding administering a development modulating treatment to a maizeplant at the desired tassel development stage.

The invention also provides a method of producing hybrid maize seed byplanting maize seed for a first parent plant in an area, growing thefirst parent plant from the maize seed, determining the timing of tasseldevelopment for the first parent plant by selecting a range thatindicates maturation to a desired tassel development stage on a RelativeDevelopment Scale, using the determination of the timing of tasseldevelopment to timely administer a development modulating treatment tothe first parent plant thereby preventing self-fertilization of thefirst parent plant, administering the development modulating treatmentto the first parent plant, fertilizing the first parent plant withpollen from a second parent plant, and harvesting seed from the firstparent plant, wherein the seed is hybrid maize seed produced by thecross of the first parent plant with the second parent plant. Theinvention also provides the hybrid maize seed produced using the method.The invention also provides a method of producing hybrid maize seed,wherein the development modulating treatment is glyphosate and the firstparent plant has tissue-selective glyphosate tolerance. The inventionalso provides a method of producing hybrid maize seed, wherein the firstparent plant is a transgenic maize event MON 87427 plant. The inventionalso provides a method of producing hybrid maize seed, wherein thesecond parent plant is glyphosate tolerant.

The foregoing and other aspects of the invention will become moreapparent from the following detailed description.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 illustrates the organization of transgenic maize event MON 87427.In the figure, [A1], [A2], and [A3] correspond to the relative positionof SEQ ID NO: 1, SEQ ID NO: 3, and SEQ ID NO: 5, respectively, whichspan the maize genomic DNA flanking the 5′ end of the transgene insertand the 5′ portion of the transgene insert DNA; [B1], [B2], and [B3]correspond to the relative position of SEQ ID NO: 2, SEQ ID NO: 4, andSEQ ID NO: 6, respectively, which span the maize genomic DNA flankingthe 3′ end of the transgene insert and the 3′ portion of the transgeneinsert DNA; [C] corresponds to the relative position of SEQ ID NO: 7,which includes the maize genomic DNA flanking the 5′ end of thetransgene insert and a portion of the 5′ end of the transgene insert;[D] corresponds to the relative position of SEQ ID NO: 8, which includesthe maize genomic DNA flanking the 3′ end of the transgene insert and aportion of the 3′ end of the transgene insert; [E] corresponds to therelative position of SEQ ID NO: 9 and the various elements in thetransgene insert; and [F] represents the contiguous sequence of MON87427 provided as SEQ ID NO: 10 and comprising SEQ ID NO: 1-9.

FIG. 2 shows the seed yield of hybrids of MON 87427 when crossed withmaize event NK603 and sprayed twice per season with glyphosate at a rateof 2.25 pounds per acre each spray.

FIG. 3 illustrates tassel development stages used in constructing theRelative Development Scale. Approximate size is shown between brackets.In the figure, Vg is meristem at vegetative stage; T0 is switch fromvegetative to reproductive; T1 is reproductive growing point visible(0.9 mm); T2 is lateral branch primordia visible (1.8 mm); T3 isspikelet primordia visible (4.1 mm); T4 is central axis and lateral axiselongation (12.9 mm); T5 is beginning of anthers differentiation (41.0mm); T6 is beginning of pollen differentiation (175 mm); and T7 isanther exertion and pollen shed (285.0 mm).

FIG. 4 illustrates tassel size variation between three maize genotypesat two developmental stages (V8 and V10).

FIG. 5 illustrates the correlation between GDU requirements for T5-Stageand those to P50% and more specifically shows the regression lineproduced using the correlation between GDU requirements to T5 and toP50%. Each dot represents a different inbred, averaged across locations.

FIG. 6 illustrates an example of how the Relative Development Scalereveals an optimal window of chemical agent efficacy for producing maizetassel sterility as measured by anther extrusion risk (AE Risk (%))which occurs at 0.62 and 0.75 on the Relative Development Scale, where62%-75% of the total GDU requirement to reach P50 are reached and inwhich AE Risk is minimized across inbreds and maturity groups. Each datapoint represents averaged values for 1 plot, or two rows totally 32plants. N=620

FIG. 7 illustrates T-stages as a function of GDU (A) and RelativeDevelopment Scale (B). Each regression line represents a differentinbred.

FIG. 8 illustrates the percentage of anther extrusion risk (y axis)measured at different silking stages (x axis) for MON 87427 and CMSblocks.

FIG. 9 illustrates the seed genetic purity and the seed trait purity ofhybrid seed produced by MON 87427 with the Roundup® Hybridization System(RHS) and by the CMS system at the 95% significance level. The blackline on the chart represents the desired quality standards for seedgenetic purity and seed trait purity, respectively.

BRIEF DESCRIPTION OF THE SEQUENCES

SEQ ID NO: 1 is a twenty nucleotide sequence representing the 5′junction region of a maize genomic DNA and an integrated transgenicexpression cassette.

SEQ ID NO: 2 is a twenty nucleotide sequence representing the 3′junction region of a maize genomic DNA and an integrated transgenicexpression cassette.

SEQ ID NO: 3 is a sixty nucleotide sequence representing the 5′ junctionregion of a maize genomic DNA and an integrated transgenic expressioncassette.

SEQ ID NO: 4 is a sixty nucleotide sequence representing the 3′ junctionregion of a maize genomic DNA and an integrated transgenic expressioncassette.

SEQ ID NO: 5 is a one-hundred nucleotide sequence representing the 5′junction region of a maize genomic DNA and an integrated transgenicexpression cassette.

SEQ ID NO: 6 is a one-hundred nucleotide sequence representing the 3′junction region of a maize genomic DNA and an integrated transgenicexpression cassette.

SEQ ID NO: 7 is the 5′ sequence flanking the inserted DNA of MON 87427up to and including a region of transgene DNA insertion.

SEQ ID NO: 8 is the 3′ sequence flanking the inserted DNA of MON 87427up to and including a region of transgene DNA insertion.

SEQ ID NO: 9 is the sequence fully integrated into the maize genomic DNAand containing the expression cassette DNA.

SEQ ID NO: 10 is the nucleotide sequence representing the contig of the5′ sequence flanking the inserted DNA of MON 87427 (SEQ ID NO: 7), thesequence fully integrated into the maize genomic DNA and containing theexpression cassette (SEQ ID NO: 9), and the 3′ sequence flanking theinserted DNA of MON 87427 (SEQ ID NO: 8) and includes SEQ ID NO: 1-6.

SEQ ID NO: 11 is transgene-specific assay Event Primer-1 SQ20052 used toidentify MON 87427. A PCR amplicon produced from a TAQMAN® (PE AppliedBiosystems, Foster City, Calif.) assay using the combination of primersSEQ ID NO: 11 and SEQ ID NO: 12 is a positive result for the presence ofthe event MON 87427.

SEQ ID NO: 12 is transgene-specific assay Event Primer-1 SQ20053 used toidentify MON 87427.

SEQ ID NO: 13 is a transgene-specific assay Event 6-FAM Probe PB10016used to identify MON 87427. This probe is a 6FAM-labeled syntheticoligonucleotide. Release of a fluorescent signal in an amplificationreaction using primers SEQ ID NO: 11-12 in combination with the6FAM™-labeled probe is diagnostic of event MON 87427 in a TAQMAN® assay.

SEQ ID NO: 14 is transgene-specific assay Internal Control Primer-1SQ1241.

SEQ ID NO: 15 is transgene-specific assay Internal Control Primer-1SQ1242.

SEQ ID NO: 16 is a transgene-specific assay Internal Control VIC ProbePB0084.

SEQ ID NO: 17 is event-specific assay Event Primer-1 SQ12763 used toidentify MON 87427. A PCR amplicon produced from a TAQMAN® (PE AppliedBiosystems, Foster City, Calif.) assay using the combination of primersSEQ ID NO: 17 and SEQ ID NO: 18 is a positive result for the presence ofthe event MON 87427.

SEQ ID NO: 18 is event-specific assay Event Primer-1 SQ12886 used toidentify MON 87427.

SEQ ID NO: 19 is a transgene-specific assay Event 6-FAM Probe PB4352used to identify MON 87427.

DETAILED DESCRIPTION

The following definitions and methods are provided to better define theinvention and to guide those of ordinary skill in the art in thepractice of the invention. Unless otherwise noted, terms are to beunderstood according to conventional usage by those of ordinary skill inthe relevant art.

As used herein, the term “maize” means “corn” or Zea mays and includesall plant varieties that can be bred with maize, including wild maizespecies as well as those plants belonging to Zea that permit breedingbetween species.

“Glyphosate” refers to N-phosphonomethylglycine, which is an herbicidethat is an enolpyruvylshikimate 3-phosphate synthase (EPSPS) inhibitor.Glyphosate interferes with the synthesis of aromatic amino acids byinhibiting EPSPS. Glyphosate is commercially available as Roundup®herbicide (Monsanto Company, St. Louis, Mo.).

The invention provides maize transgenic event MON 87427 (also referredto herein as MON 87427). As used herein, the term “event” refers to aproduct created by the act of inserting a transgenic nucleic acidmolecule into the genome of a plant, i.e., by the act of planttransformation to produce a transgenic plant. An “event” is thereforeproduced by the human acts of: (i) transforming a plant cell in alaboratory with a nucleic acid molecule that includes a transgene ofinterest, i.e., inserting into the plant cell's genome the nucleic acidconstruct or molecule, (ii) regenerating a population of transgenicplants resulting from the insertion of the nucleic acid molecule intothe genome of the plant, and (iii) selecting a particular plantcharacterized by the insertion of the nucleic acid molecule into aparticular location in the plant's genome. The event may therefore beuniquely and specifically described by a nucleic acid sequencerepresenting at least a portion of the contiguous DNA molecule that wasproduced in the event by the insertion of the nucleic acid molecule intothe particular location in the plant's genome and that includes aportion of the plant's own genomic DNA, which flanks and is physicallyconnected to the inserted DNA molecule, and the inserted nucleic acidmolecule. An event is recombinant, produced by human actions, and notfound in nontransgenic plants.

The term “event” thus refers to the original transformed plant(“transformant”) that includes the nucleic acid molecule inserted intothe particular location in the plant's genome. The term “event” alsorefers to all progeny descended from the transformant that include thenucleic acid molecule inserted into the particular location in theplant's genome. Such progeny are consequently transgenic and comprisethe event. Progeny may be produced by any means includingself-fertilization, crossing with another plant that comprises the sameor different transgene, and/or crossing with a nontransgenic plant, suchas one from a different variety. Even after many generations, in anyplant referred to as a MON 87427 progeny plant the inserted DNA and theflanking DNA from the original transformed plant will be present andreadily identifiable.

The term “event” also refers to the contiguous DNA molecule created inthe original transformant (comprising the inserted DNA and the flankingmaize genomic DNA immediately adjacent to either side of the insertedDNA) or any DNA molecule comprising that nucleic acid sequence. Thecontiguous DNA molecule was created by the act of inserting a transgenicnucleic acid molecule into the genome of a plant, i.e., by the act oftransformation, and is specific and unique to the particular event. Thearrangement of the inserted DNA in MON 87427 in relation to thesurrounding maize plant genomic DNA is therefore specific and unique forMON 87427. This DNA molecule is also an integral part of the maizechromosome of MON 87427 and as such is static in the plant and may beinherited by any progeny.

Transgenic maize event MON 87427 plants exhibit commercially acceptabletissue-selective glyphosate tolerance. In MON 87427, the maizevegetative tissues and the maize female reproductive tissues areglyphosate tolerant, but key maize male reproductive tissues criticalfor maize pollen development are not glyphosate tolerant.Glyphosate-treated MON 87427 plants may therefore be used as a femaleparent in the production of hybrid seed.

As used herein, the term “recombinant” refers to a non-natural DNAand/or protein and/or an organism that would not normally be found innature and was created by human intervention, i.e., by human hands. Suchhuman intervention may produce a DNA molecule and/or a plant or seed. Asused herein, a “recombinant DNA molecule” is a DNA molecule comprising acombination of DNA molecules that would not naturally occur together andis the result of human intervention, e.g., a DNA molecule that iscomprised of a combination of at least two DNA molecules heterologous toeach other, and/or a DNA molecule that is artificially synthesized andhas a nucleotide sequence that deviates from the nucleotide sequencethat would normally exist in nature, and/or a DNA molecule thatcomprises a nucleic acid molecule artificially incorporated into a hostcell's genomic DNA and the associated flanking DNA of the host cell'sgenome. An example of a recombinant DNA molecule is a DNA moleculecomprising at least one sequence selected from SEQ ID NO: 1-10. As usedherein, a “recombinant plant” is a plant that would not normally existin nature, is the result of human intervention, and contains a transgeneand/or heterologous DNA molecule incorporated into its genome. As aresult of such genomic alteration, the recombinant plant is distinctlydifferent from the related wildtype plant. An example of a recombinantplant is a transgenic maize event MON 87427 plant.

As used herein, the term “transgene” refers to a nucleic acid moleculeartificially incorporated into an organism's genome as a result of humanintervention. Such transgene may be heterologous to the host cell. Theterm “transgenic” refers to comprising a transgene, for example a“transgenic plant” refers to a plant comprising a transgene, i.e., anucleic acid molecule artificially incorporated into the organism'sgenome as a result of human intervention. As used herein, the term“heterologous” refers to a first molecule not normally found incombination with a second molecule in nature. For example, a moleculemay be derived from a first species and inserted into the genome of asecond species. The molecule would thus be a heterologous molecule,i.e., heterologous to the organism and artificially incorporated intothe organism's genome.

As used herein, the term “chimeric” refers to a single DNA moleculeproduced by fusing a first DNA molecule to a second DNA molecule, whereneither first nor second DNA molecule would normally be found in thatconfiguration, i.e., fused to the other. The chimeric DNA molecule isthus a new DNA molecule not otherwise normally found in nature. Anexample of a chimeric DNA molecule is a DNA molecule comprising at leastone sequence selected from SEQ ID NO: 1-10.

The invention provides DNA molecules and their corresponding nucleotidesequences. As used herein, the term “DNA”, “DNA molecule”, “nucleic acidmolecule” refers to a double-stranded DNA molecule of genomic orsynthetic origin, i.e., a polymer of deoxyribonucleotide bases or anucleotide molecule, read from the 5′ (upstream) end to the 3′(downstream) end. As used herein, the term “DNA sequence” or “nucleotidesequence” refers to the nucleotide sequence of a DNA molecule. Thenomenclature used herein is that required by Title 37 of the UnitedStates Code of Federal Regulations § 1.822 and set forth in the tablesin WIPO Standard ST.25 (1998), Appendix 2, Tables 1 and 3. By conventionand as used herein, the nucleotide sequences of the invention, such asthose provided as SEQ ID NO: 1-10 and fragments thereof, are providedwith reference to only one strand of the two complementary nucleotidesequence strands, but by implication the complementary sequences (i.e.the sequences of the complementary strand), also referred to in the artas the reverse complementary sequences, are within the scope of theinvention and are expressly intended to be within the scope of thesubject matter claimed. Thus, as used herein references to SEQ ID NO:1-10 and fragments thereof include and refer to the sequence of thecomplementary strand and fragments thereof.

As used herein, the term “fragment” refers to a portion of or anincomplete smaller piece of a whole. For example, fragments of SEQ IDNO: 10 would include sequences that are at least about 10 nucleotides,at least about 20 nucleotides, or at least about 50 nucleotides of thecomplete sequence of SEQ ID NO: 10.

The nucleotide sequence corresponding to the complete nucleotidesequence of the inserted transgenic DNA and substantial segments of themaize genome DNA flanking either end of the inserted transgenic DNA isprovided herein as SEQ ID NO: 10. A subsection of this is the insertedtransgenic DNA (also referred to herein as the transgene insert or theinserted DNA) provided as SEQ ID NO: 9. The nucleotide sequence of themaize genomic DNA physically linked by phosphodiester bond linkage toand therefore flanking the 5′ end of the inserted transgenic DNA, andcontaining 10 nt of the transgene inserted DNA, is set forth as shown inSEQ ID NO: 7. The nucleotide sequence of the maize genome DNA physicallylinked by phosphodiester bond linkage to and therefore flanking the 3′end of the inserted transgenic DNA, and containing 10 nt of thetransgene inserted DNA, is set forth as shown in SEQ ID NO: 8.

The MON 87427 further comprises two regions referred to as junctions. A“junction” is where one end of the inserted transgenic DNA has beeninserted into and connected to the genomic DNA. A junction spans, i.e.,extends across, a portion of the inserted transgenic DNA and theadjacent flanking genomic DNA and as such comprises the connection pointof these two as one contiguous molecule. One junction is at the 5′ endof the inserted transgenic DNA and one is at the 3′ end of the insertedtransgenic DNA, referred to herein as the 5′ and 3′ junction,respectively. A “junction sequence” or “junction region” refers to theDNA sequence and/or corresponding DNA molecule of the junction. Junctionsequences of MON 87427 can be designed by one of skill in the art usingSEQ ID NO: 10. Examples of junction sequences of MON 87427 are providedas SEQ ID NO: 1-6. SEQ ID NO: 1 is a 20 nucleotide sequence spanning thejunction between the maize genomic DNA and the 5′ end of the transgenicinsert DNA; SEQ ID NO: 3 is a 60 nucleotide sequence spanning thejunction between the maize genomic DNA and the 5′ end of the transgenicinsert DNA; SEQ ID NO: 5 is a 100 nucleotide sequence spanning thejunction between the maize genomic DNA and the 5′ end of the transgenicinsert DNA. SEQ ID NO: 2 is a 20 nucleotide sequence spanning thejunction between the maize genomic DNA and the 3′ end of the insertedDNA; SEQ ID NO: 4 is a 60 nucleotide sequence spanning the junctionbetween the maize genomic DNA and the 3′ end of the inserted DNA; SEQ IDNO: 6 is a 100 nucleotide sequence spanning the junction between themaize genomic DNA and the 3′ end of the inserted DNA. FIG. 1 illustratesthe physical arrangement of SEQ ID NO: 1-10 arranged from 5′ to 3′. Anysegment of DNA derived from transgenic MON 87427 that includes SEQ IDNO: 1-6 is within the scope of the invention. The invention thusprovides a DNA molecule that contains at least one of the nucleotidesequences as set forth in SEQ ID NO: 1-6.

The junction sequences of event MON 87427 are present as part of thegenome of a transgenic maize event MON 87427 plant, seed, or cell. Theidentification of any one or more of SEQ ID NO: 1-6 in a sample derivedfrom a maize plant, seed, or plant part is determinative that the DNAwas obtained MON 87427 and is diagnostic for the presence in a sample ofDNA from MON 87427.

The invention provides exemplary DNA molecules that can be used eitheras primers or probes for diagnosing the presence of DNA derived frommaize plant event MON 87427 in a sample. Such primers or probes arespecific for a target nucleic acid sequence and as such are useful forthe identification of MON 87427 nucleic acid sequences by the methods ofthe invention described herein.

A “primer” is a nucleic acid molecule that is designed for use inannealing or hybridization methods that involve thermal amplification. Apair of primers may be used with template DNA, such as a sample of maizegenomic DNA, in a thermal amplification, such as polymerase chainreaction (PCR), to produce an amplicon, where the amplicon produced fromsuch reaction would have a DNA sequence corresponding to sequence of thetemplate DNA located between the two sites where the primers hybridizedto the template. As used herein, an “amplicon” is DNA that has beensynthesized using amplification techniques. Amplicons of the inventionhave a sequence comprising one or more of SEQ ID NO: 1-10 or fragmentsthereof. A primer is typically designed to hybridize to a complementarytarget DNA strand to form a hybrid between the primer and the target DNAstrand, and the presence of the primer is a point of recognition by apolymerase to begin extension of the primer (i.e., polymerization ofadditional nucleotides into a lengthening nucleotide molecule) using asa template the target DNA strand. Primer pairs, as used in theinvention, are intended to refer to use of two primers binding oppositestrands of a double stranded nucleotide segment for the purpose ofamplifying linearly the nucleotide segment between the positionstargeted for binding by the individual members of the primer pair.Examples of primer sequences are provided as SEQ ID NO: 11-12, SEQ IDNO: 14-15 and SEQ ID NO: 17-18. The primer pair of SEQ ID NO: 14-15 andthe primer pair of SEQ ID NO: 17-18 each have a first DNA molecule and asecond DNA molecule (that is different from the first DNA molecule)where both molecules are each of sufficient length of contiguousnucleotides to function as DNA primers that, when used together in a DNAamplification reaction with template DNA derived from MON 87427, producean amplicon diagnostic for the presence in a sample of DNA from MON87427.

A “probe” is a nucleic acid molecule that is complementary to a strandof a target nucleic acid for use in annealing or hybridization methods.Probes according to the invention include not only deoxyribonucleic orribonucleic acids but also polyamides and other probe materials thatbind specifically to a target DNA sequence and the detection of suchbinding can be useful in diagnosing, discriminating, determining, orconfirming the presence of that target DNA sequence in a particularsample. A probe may be attached to a conventional detectable label orreporter molecule, e.g., a radioactive isotope, ligand, chemiluminescentagent, or enzyme. Examples of sequences useful as probes for detectingMON 87427 are SEQ ID NO: 1-2, SEQ ID NO: 13, SEQ ID NO: 16, SEQ ID NO:19.

Methods for designing and using primers and probes are well know in theart and are described by, for example, Joseph Sambrook, MolecularCloning: A Laboratory Manual, Third Edition, Cold Spring HarborLaboratory Press (2001) and Current Protocols in Molecular Biology,Wiley-Blackwell. DNA molecules comprising fragments of SEQ ID NO: 1-10and useful as primers and probes for detecting MON 87427 can readily bedesigned by one of skill in the art for use as probes in hybridizationdetection methods, e.g., Southern Blot method

The DNA molecules and corresponding nucleotide sequences provided hereinare therefore useful for, among other things, identifying MON 87427,selecting plant varieties or hybrids comprising MON 87427, detecting thepresence of DNA derived from the transgenic MON 87427 in a sample, andmonitoring samples for the presence and/or absence of MON 87427 or plantparts derived from MON 87427.

The invention provides maize plants, progeny, seeds, plant cells, plantparts, and commodity products. These plants, progeny, seeds, plantcells, plant parts, and commodity products contain a detectable amountof a nucleotide of the invention, i.e., such as a nucleic acid moleculecomprising at least one of the sequences provided as SEQ ID NO: 1-10.Plants, progeny, seeds, plant cells, and plant parts of the inventionmay also contain one or more additional transgenes. Such transgene maybe any nucleotide sequence encoding a protein or RNA molecule conferringa desirable trait including but not limited to increased insectresistance, increased water use efficiency, increased yield performance,increased drought resistance, increased seed quality, improvednutritional quality, and/or increased herbicide tolerance, in which thedesirable trait is measured with respect to a maize plant lacking suchadditional transgene.

The invention provides maize plants, progeny, seeds, plant cells, andplant parts, and leaves derived from a transgenic maize plant event MON87427. A representative sample of MON 87427 seed has been depositedaccording to the Budapest Treaty for the purpose of enabling theinvention. The repository selected for receiving the deposit is theAmerican Type Culture Collection (ATCC) having an address at 10801University Boulevard, Manassas, Va. USA, Zip Code 20110. The ATCCrepository has assigned the accession No. PTA-7899 to the event MON87427 seed.

The invention provides a microorganism comprising a DNA molecule havingSEQ ID NO: 1-10 present in its genome. An example of such amicroorganism is a transgenic plant cell. Microorganisms, such as aplant cell of the invention, are useful in many industrial applications,including but not limited to: (i) use as research tool for scientificinquiry or industrial research; (ii) use in culture for producingendogenous or recombinant carbohydrate, lipid, nucleic acid, or proteinproducts or small molecules that may be used for subsequent scientificresearch or as industrial products; and (iii) use with modern planttissue culture techniques to produce transgenic plants or plant tissuecultures that may then be used for agricultural research or production.The production and use of microorganisms such as transgenic plant cellsutilizes modern microbiological techniques and human intervention toproduce a man-made, unique microorganism. In this process, recombinantDNA is inserted into a plant cell's genome to create a transgenic plantcell that is separate and unique from naturally occurring plant cells.This transgenic plant cell can then be cultured much like bacteria andyeast cells using modern microbiology techniques and may exist in anundifferentiated, unicellular state. The new plant cell's geneticcomposition and phenotype is a technical effect created by theintegration of the heterologous DNA into the genome of the cell. Anotheraspect of the invention is a method of using a microorganism of theinvention. Methods of using microorganisms of the invention, such astransgenic plant cells, include (i) methods of producing transgeniccells by integrating recombinant DNA into genome of the cell and thenusing this cell to derive additional cells possessing the sameheterologous DNA; (ii) methods of culturing cells that containrecombinant DNA using modern microbiology techniques; (iii) methods ofproducing and purifying endogenous or recombinant carbohydrate, lipid,nucleic acid, or protein products from cultured cells; and (iv) methodsof using modern plant tissue culture techniques with transgenic plantcells to produce transgenic plants or transgenic plant tissue cultures.

Plants of the invention may pass along the event DNA, including thetransgene insert, to progeny. As used herein, “progeny” includes anyplant, seed, plant cell, and/or regenerable plant part comprising theevent DNA derived from an ancestor plant and/or a nucleic acid moleculehaving at least one of the sequences provided as SEQ ID NO: 1-10.Plants, progeny, and seeds may be homozygous or heterozygous for thetransgene. Progeny may be grown from seeds produced by a MON 87427 plantand/or from seeds produced by a plant fertilized with pollen from a MON87427 plant. Plants of the invention may be produced by self-pollinationor cross-pollination and/or may be used in self-pollination orcross-pollination methods. Thus in one embodiment, a MON 87427 plant canbe cross-pollinated by a different maize plant to produce hybridoffspring. Breeding methods useful with MON 87427 maize plants are knownin the art.

The invention provides a plant part that is derived from MON 87427. Asused herein, a “plant part” refers to any part of a plant which iscomprised of material derived from a MON 87427 plant. Plant partsinclude but are not limited to pollen, ovule, pod, flower, root or stemtissue, fibers, and leaves. Plant parts may be viable, nonviable,regenerable, and/or nonregenerable.

The invention provides a commodity product that is produced fromtransgenic maize event MON 87427 and comprises a nucleic acid moleculehaving a nucleotide sequence selected from the group consisting of SEQID NO: 1-10. As used herein, a “commodity product” refers to anycomposition or product which is comprised of material derived from a MON87427 plant, seed, plant cell, or plant part. Commodity products may beviable or nonviable. Nonviable commodity products include but are notlimited to nonviable seeds and grains; processed seeds, seed parts, andplant parts; dehydrated plant tissue, frozen plant tissue, and processedplant tissue; seeds and plant parts processed for animal feed forterrestrial and/or aquatic animals consumption, oil, meal, flour,flakes, bran, fiber, milk, cheese, paper, cream, wine, and any otherfood for human consumption; and biomasses and fuel products. Viablecommodity products include but are not limited to seeds and plant cells.Transgenic maize event MON 87427 can thus be used to manufacture anycommodity product typically acquired from maize. A commodity productthat is derived from the MON 87427 may contain a detectable amount ofthe specific and unique DNA corresponding to MON 87427, and specificallymay contain a detectable amount of a nucleic acid molecule having atleast one of the sequences provided as SEQ ID NO: 1-10. Detection of oneor more of these sequences in a sample of a commodity product derivedfrom, made up of, consisting of, or comprising a corn plant, a cornseed, a corn plant cell, or a corn plant part is conclusive anddeterminative of the presence of biological material derived from cornevent MON87427 in such commodity product, and the detection of such anucleic acid molecule may be used for determining the content and/or thesource of the commodity product. Any standard method of detection fornucleic acid molecules may be used, including methods of detectiondisclosed herein.

The plants, progeny, seeds, plant cells, plant parts, and commodityproducts of the invention are therefore useful for, among other things,growing plants for the purpose of producing seed and/or plant parts ofMON 87427 for agricultural purposes, producing progeny of MON 87427 forplant breeding and research purposes, use with microbiologicaltechniques for industrial and research applications, and sale toconsumers.

The invention provides methods for controlling weeds using glyphosateherbicide and MON 87427. A method for controlling weeds in a field isprovided and consists of planting MON 87427 varietal or hybrid plants ina field and applying an herbicidally effective dose of glyphosate to thefield for the purpose of controlling weeds in the field without injuringthe MON 87427 plants. Such application of glyphosate herbicide may bepre-emergence, i.e., any time after MON 87427 seed is planted and beforeMON 87427 plants emerge, or post-emergence, i.e., any time after MON87427 plants emerge. An herbicidally effective dose of glyphosate foruse in the field for controlling weeds should consist of a range fromabout 0.1 pound per acre to as much as about 4 pounds of glyphosate peracre over a growing season. Multiple applications of glyphosate may beused over a growing season, for example, two applications (such as apre-planting application and a post-emergence application or apre-emergence application and a post-emergence application) or threeapplications (such as a pre-planting application, a pre-emergenceapplication, and a post-emergence application).

Methods for producing an herbicide tolerant transgenic maize event MON87427 plant are provided. Progeny produced by these methods may bevarietal or hybrid plants; may be grown from seeds produced by a MON87427 plant and/or from seeds produced by a plant fertilized with pollenfrom a MON 87427 plant; and may be homozygous or heterozygous for thetransgene. Progeny may be subsequently self-pollinated orcross-pollinated.

A maize plant that tolerates application of glyphosate herbicide may beproduced by sexually crossing a MON 87427 plant comprising a nucleicacid molecule comprising at least one of the sequences provided as SEQID NO: 1-10 with another maize plant and thereby producing seed, whichis then collected and grown into progeny plants. These progeny may thenbe treated with glyphosate herbicide and progeny that are tolerant toglyphosate herbicide may be selected. Alternatively, these progenyplants may be analyzed using diagnostic methods to select for progenyplants that contain MON 87427 DNA.

In practicing the methods of the invention, the step of sexuallycrossing one plant with another plant, i.e., cross-pollinating may beaccomplished or facilitated by human intervention, for example: by humanhands collecting the pollen of one plant and contacting this pollen withthe style or stigma of a second plant and then optionally preventingfurther fertilization of the fertilized plant; by human hands and/oractions removing (e.g., by detasseling), destroying (e.g., by use ofchemical agents), or covering the stamen or anthers of a plant so thatnatural self-pollination is prevented and cross-pollination would haveto take place in order for fertilization to occur; by human placement ofpollinating insects in a position for “directed pollination” (e.g., byplacing beehives in orchards or fields or by caging plants withpollinating insects); by human opening or removing of parts of theflower to allow for placement or contact of foreign pollen on the styleor stigma (e.g., in maize which naturally has flowers that hinder orprevent cross-pollination, making them naturally obligateself-pollinators without human intervention); by selective placement ofplants in a specific area (e.g., intentionally planting plants inpollinating proximity); and/or by application of chemicals toprecipitate flowering or to foster receptivity (of the stigma forpollen).

In practicing the methods of the invention, the step of sexuallyfertilizing a maize plant by self-pollination, i.e., selfing, may beaccomplished or facilitated by human intervention, for example: by humanhands collecting the pollen of a plant and contacting this pollen withthe style or stigma of the same plant and then optionally preventingfurther fertilization of the fertilized plant; by human hands and/oractions removing (e.g., by detasseling), destroying (e.g., by use ofchemical agents), or covering the stamen or anthers of a plant so thatnatural self-pollination is prevented and manual self-pollination wouldhave to take place in order for fertilization to occur; by humanplacement of pollinating insects in a position for “directedpollination” (e.g., by caging a plant alone with pollinating insects);by human manipulation of a plant's reproductive parts to allow for orenhance self-pollination; by selective placement of plants (e.g.,intentionally planting other plants beyond pollinating proximity);and/or by application of chemicals to precipitate flowering or to fosterreceptivity (of the stigma for pollen).

The invention provides plants and methods useful in hybrid maize seedproduction.

The maize plant has separate male and female flowering parts. The tasselis the male structure and the ear shoot is the female floweringstructure of the plant. The flowering stage in maize involves pollenshed and silking. Maize pollen may fertilize the same plant(self-pollination) or a different plant (cross-pollination). If the malestructures of the plant are not removed prior to pollen shed, then themaize plant will self-pollinate to some extent. For hybrid seedproduction, the female structures of a first maize plant arecross-pollinated with the pollen from a second maize plant. Efficienthybrid seed production thus requires that a plant's own pollen not bepermitted to self-fertilize the plant. Methods to enhance hybrid maizeseed production provided herein comprise growing in an area a seed orplant comprising MON 87427 and one or more other maize plant(s). Theevent MON 87427 plants are then treated with glyphosate prior to pollenformation, thereby making the event MON 87427 plants male sterile andincapable of self-fertilization. The event MON 87427 plants are thenpollinated by pollen from the other maize plant(s) using any of themethods described herein. The other maize plant(s) may or may not beglyphosate tolerant. Maize seed is then harvested from the event MON87427 plants, wherein the seed harvested from the treated MON 87427plants has a higher yield of hybrid maize seed (i.e. higher percentageof hybrid seed harvested or higher hybrid seed purity) than maize seedharvested from untreated event MON 87427 plants or from other maizeplant(s) under the same conditions. The maize seed harvested fromuntreated event MON 87427 plants under the same conditions would have ahigher percentage of non-hybrid seed (i.e., inbred seed produced byself-pollination) and thus a lower yield of hybrid maize seed.

The plants and methods of the invention may also be used for maizebreeding purposes with methods known in the art including using themethods described in U.S. Pat. No. 7,314,970, which is herebyincorporated by reference herein, and U.S. Patent Publication No.20090165166, which is hereby incorporated by reference herein.

Plants, progeny, and seeds encompassed by these methods and produced byusing these methods will be distinct from other maize plants. Forexample the MON 87427 plants, progeny, and seeds of the invention aretransgenic and recombinant and as such created by human intervention andcontain a detectable amount of a nucleic acid molecule having at leastone of the sequences provided as SEQ ID NO: 1-10.

The methods of the invention are therefore useful for, among otherthings, controlling weeds in a field while growing plants for thepurpose of producing seed and/or plant parts of MON 87427 foragricultural or research purposes, selecting for progeny of MON 87427for plant breeding or research purposes, and producing progeny plantsand seeds of MON 87427.

The plants, progeny, seeds, plant cells, plant parts, and commodityproducts of the invention may be evaluated for DNA composition, geneexpression, and/or protein expression. Such evaluation may be done byusing standard methods such as PCR, northern blotting, southernanalysis, western blotting, immuno-precipitation, and ELISA or by usingthe methods of detection and/or the detection kits provided herein.

Methods of detecting the presence of materials specific to MON 87427 ina sample are provided. It is possible to detect the presence of anucleic acid molecule of the invention by using the probes and primersof the invention with any nucleic acid detection method used in the art,such as polymerase chain reaction (PCR) or DNA hybridization. One methodprovides for contacting a DNA sample with a primer pair that is capableof producing an amplicon from event MON 87427 DNA, performing anamplification reaction and thereby producing a DNA amplicon comprisingat least one of the nucleotide sequences provided as SEQ ID NO: 1-10,and then detecting the presence or absence of the amplicon molecule andoptionally confirming within the sequence of the amplicon a sequencecomprising to at least one of the sequences provided as SEQ ID NO: 1-10.The presence of such an amplicon is determinative and/or diagnostic forthe presence of the MON 87427 specific DNA and thus MON 87427 biologicalmaterial in the sample. Another method provides for contacting a DNAsample with a DNA probe, subjecting the probe and the DNA sample tostringent hybridization conditions, and then detecting hybridizationbetween the probe and the target DNA sample. Detection of hybridizationis diagnostic for the presence of MON 87427 specific DNA in the DNAsample. Nucleic-acid amplification, nucleic acid hybridization, and DNAsequencing can be accomplished by any of the methods known in the art.One exemplary technique useful in practicing this invention is TAQMAN®(PE Applied Biosystems, Foster City, Calif.).

The sequence of the heterologous DNA insert, junction sequences, orflanking sequences from MON 87427 (with representative seed samplesdeposited as ATCC PTA-7899) can be verified (and corrected if necessary)by amplifying such sequences from the event using primers derived fromthe sequences provided herein followed by standard DNA sequencing of theamplicon or of the cloned DNA.

DNA detection kits are provided. Variations on such kits can also bedeveloped using the compositions and methods disclosed herein and themethods well known in the art of DNA detection. DNA detection kits areuseful for the identification of MON 87427 DNA in a sample and can beapplied to methods for breeding maize plants containing MON 87427 DNA.The kits may contain DNA primers or probes that are similar orcomplementary to SEQ ID NO: 1-10 or fragments thereof.

The kits and detection methods of the invention are therefore usefulfor, among other things, identifying MON 87427, selecting plantvarieties or hybrids comprising MON 87427, detecting the presence of DNAderived from the transgenic MON 87427 in a sample, and monitoringsamples for the presence and/or absence of MON 87427 or plant partsderived from MON 87427.

The invention provides a Relative Development Scale useful formonitoring and/or determining reproductive development in maize. Thisnovel Relative Development Scale resolves the developmental andreproductive maturation differences across various maize varieties andinbreds by providing a time scale that expresses tassel developmentstages relative to flowering. The Relative Development Scale diminishesthe observed differences in tassel development and tassel growth acrossgenotypes. Tassel development in the various stages of maturation isillustrated in FIG. 3.

Maize development is often determined by a scale of stages based onvegetative events, commonly known as V-Stages. These stages are definedaccording to the uppermost leaf in which the leaf collar is visible. VEcorresponds to emergence, V1 corresponds to first leaf, V2 correspondsto second leaf, V3 corresponds to third leaf, V(n) corresponds to nthleaf. VT occurs when the last branch of tassel is visible but beforesilks emerge. When staging a field of maize, each specific V-stage isdefined only when 50 percent or more of the plants in the field are inor beyond that stage. However, the use of this vegetative scale todetermine reproductive maturity may be complicated by the fact thatvegetative development does not necessarily correlate to reproductivedevelopment across all genotypes. In addition, not all inbredsdifferentiate the same number of leaves, field inspectors are not alwaysconsistent in their assessment, and the first leaves to differentiatestart to senesce fairly early in the season so if leaves are notproperly marked during the early stages it becomes very difficult toproperly identify the V-stages later on.

Another common tool for predicting and estimating stages of maize growthand development is Growing Degree Units (GDU). A factor in the growthand development of maize is heat. Heat is typically measured at a singlepoint in time and is expressed as temperature, but it can also bemeasured over a period of time and be expressed as heat units. Theseheat units are commonly referred to as GDU's. GDU's may be defined asthe difference between the average daily temperature and a selected basetemperature subject to certain restrictions. GDU's are calculated usingthe following equation:

Growing Degree Unit={(H+L)/2}−B

where H is the daily high (but no higher than 86° F.), L is the dailylow (but no lower than 50° F.), and B is the base of 50° F. Becausemaize growth is minor when temperatures are greater than 86° F. or lessthan 50° F., limits are set on the daily high and low temperatures usedin the formula. The lower cutoff for daily temperature also preventscalculation of negative values. Therefore, if the daily high temperatureexceeds 86° F., the daily high temperature used in the GDU formula wouldbe set at 86° F. Conversely, if the daily low temperature drops below50° F., the daily low temperature used in the GDU formula would be setat 50° F. If the daily high temperature does not exceed 50° F., then noGDU is recorded for that day. The maximum GDU a maize plant canaccumulate in a day is 36, the minimum is zero. A maize plant's maturityrating is identified by the sum of the daily GDU values over a specifiedamount of time. The time period that most maize seed producers use isfrom the point of planting to physiological maturity or the point atwhich grain fill is virtually complete. In most U.S. states, accumulatedGDU's are kept for most geographic areas and are available from the USDACrop Reporting Service or the State Extension Services. Additionally, aninstrument for obtaining GDU information at a particular location isalso described in U.S. Pat. No. 6,967,656, which is hereby incorporatedby reference in its entirety herein. As with V-Stages, GDU measurementsmay vary significantly relative to tassel development stage acrossgenotypes and may not be a reliable predictor of tassel development.

As used herein, a “Relative Development Scale” is defined as a scalecreated by dividing the GDU at a given tassel development stage by theGDU required to achieve a particular stage of pollen shed. A regressionline is then constructed with this information for each genotype orinbred variety. A Relative Development Scale may be constructed usingthe methods described herein and is based on the correlation between theGDU requirements necessary to reach a certain maize flower developmentstage relative to a given tassel development stage. As such, a RelativeDevelopment Scale is useful for predicting tassel development in maizeacross various genotypes and inbred varieties and may be used as analternative to using V-Stages or GDU's in plant breeding andagricultural methods.

As used herein, “flower development stage” is defined according to theextent to which a population of plants is shedding pollen, referred toas P-Stage. Flower development stage is expressed as Px, where P standsfor “pollen” and “x” indicates the percentage of plants within apopulation that are shedding pollen. The Relative Development Scale ofthe invention is based on a regression derived by dividing GDUs at agiven tassel development stage by the number of GDUs required to achievea particular stage of pollen shed. This is expressed as follows:

Relative Development Scale=(GDU to Tn/GDU to Px)

where “GDU to Tn” is the amount of GDU (growing degree units) requiredto a reach a certain tassel development stage where n could range from 0to 7, and where “GDU to Px” is the amount of GDU required to reach acertain flower development stage or P-Stage where x can range from 0 to100 (an example of this is P50 defined as 50% of the plants in the fieldhave started shedding pollen).

The regression may be based on the correlative relationship between anytassel development stage and flower development stage or P-Stage GDUrequirements. Such correlative relationship is expressed by dividing theGDU required to reach a specified tassel development stage by the GDUrequired to reach a specified flower development stage or P-Stage. Inone embodiment of the invention, the flower development stage or P-Stagefor the regression is P50, wherein 50% of a population of the maizeplants is shedding pollen. In another embodiment, the flower developmentstage or P-Stage of pollen shed for calculating the regression may befrom about 1% to 100%, including about 2, 3, 4, 5, 6, 7, 8, 9, 10, 11,12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29,30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47,48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65,66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83,84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, and 99%. Thetassel development stage for the regression may be between T0 and T7,such as T0, T1, T2, T3, T4, T5, T6, and T7. Regardless of tasseldevelopment stage and flower development stage chosen for creating theregression, a Relative Development Scale may be derived by plotting therelationship of GDUs required to achieve a particular stage of pollenshed relative to the number of GDUs required to achieve a given tasseldevelopment stage. This aspect is illustrated in FIG. 5.

As used herein, the term “determining” refers to the act of measuring,assessing, evaluating, estimating, monitoring, and/or predicting. Forexample, “determining tassel development” as used herein includesmeasuring the current stage of tassel development, monitoring theprogression of tassel development, and/or predicting the occurrence of afuture stage of tassel development.

The invention therefore provides a method for producing a RelativeDevelopment Scale comprising measuring the Growing Degree Units requiredfor a population of maize plants to mature to a specific tasseldevelopment stage; measuring the Growing Degree Units required for saidpopulation of maize plants to mature to a specific flower developmentstage; and creating a regression line by dividing said measured GrowingDegree Units required for said population of maize plants to mature tosaid specified tassel development stage by said measured Growing DegreeUnits required for said population of maize plants to mature to saidspecified flower development stage. The measuring step may be repeatedfor at least two populations of maize plants. The measuring step may berepeated for multiple tassel development stages and/or multiple flowerdevelopment stages. The specific flower development stage may be pollenshed for about 50 percent of the population of maize plants.

The invention also provides a method for determining the optimal rangewithin the Relative Development Scale for a treatment regimen linked totassel development, thus allowing one to determine the optimal timing ofa treatment regimen in which development stage is an important factor.An example of this is the application of a single common chemical agenttreatment schedule for maximum efficacy in causing maize tasselsterility across diverse parent genotypes in the production of hybridmaize seed, regardless of genotype or maturity group.

As used herein, the term “hybrid seed” is seed produced bycross-pollinating two plants. Plants grown from hybrid seed may haveimproved agricultural characteristics, such as better yield, greateruniformity, and/or disease resistance. Hybrid seed does not breed true,i.e., the seed produced by self-fertilizing a hybrid plant (the plantgrown from a hybrid seed) does not reliably result the next generationin an identical hybrid plant. Therefore, new hybrid seed must beproduced from the parent plant lines for each planting. Since most cropplants have both male and female organs, hybrid seed can only beproduced by preventing self-pollination of the female parent andallowing or facilitating pollination with the desired pollen. There area variety of methods to prevent self-pollination of the female parent,one method by which self-pollination is prevented is mechanical removalof the pollen producing organ before pollen shed. Commercial hybridmaize seed (maize, Zea mays) production typically involves planting thedesired male and female parental lines, usually in separate rows orblocks in an isolated field, treating the female parent plant to preventpollen shed, ensuring pollination of the female by only the designatedmale parent, and harvesting hybrid seed from only the female parent.Hybrid seed may be the result of a single cross (e.g., a firstgeneration cross between two inbred lines), a modified single cross(e.g., a first generation cross between two inbred lines, one or otherof which may have been modified slightly by the use of closely relatedcrossing), a double cross (e.g., a first generation of a cross betweentwo single crosses), a three-way cross (e.g., a first generation of across between a single cross and an inbred line), a top cross (e.g., thefirst generation of a cross between an inbred line and anopen-pollinated variety, or the first generation of a cross between asingle-cross and an open-pollinated variety), or an open pollinatedvariety (e.g., a population of plants selected to a standard which mayshow variation but has characteristics by which a variety can bedifferentiated from other varieties).

In hybrid seed production, pollen production and/or shed may beprevented in a female parent plant in order to facilitate pollination ofthe female by only the designated male parent and thus produce hybridseed. Such prevention may be achieved by any method or means known tothose of skill in the art, including but not limited to, manual or handdetasseling, mechanical detasseling, use of a genetic means ofpollination control, and/or use of a chemical agent. Any of these may becombined or used individually. Detasseling may be done manually or byhand and is typically performed by a person removing the tassels from amaize plant, usually by pulling the tassel off. Mechanical or machinedetasseling typically utilizes a detasseling machine called a “cutter”that moves through rows of maize and cuts off the top portion of theplant. A “puller” machine then moves through the maize rows a few dayslater and pulls the tassel out of the plant by catching it between tworollers moving at a high speed. Mechanical detasselers useful inpracticing the methods of the invention include those mounted on highclearance machines. The cutter may be a rotating blade or knife thatoperates at various planes from horizontal to vertical, adjustable inheight, to cut or shred the top of the maize plant including the tassel.The puller may be two small wheels or rollers, adjustable in height,that rotate in opposite directions and grasp the tassel and upperleaves, pulling them upward in a manner comparable to a hand detasselingoperation. Pullers and cutters may be used separately or together and/orin combination with other detasseling methods. The window of time fordetasseling is usually the most critical and difficult to manage periodin hybrid maize seed production. In the art, chemical agents and/orgenetic means are also used to prevent viable pollen formation or pollenshed.

The invention provides a method for determining the timing of tasseldevelopment by selecting a range on a Relative Development Scale,wherein the selected range indicates maturation to a desired tasseldevelopment stage. The desired tassel development stage is from the T0to the T7 stage, for example, the T5 stage. Tassel development stages ofparticular interest are the optimal tassel development stage forreproductive crossing, the optimal tassel development stage for tasselsterilization or detasseling, and/or the optimal stage foradministration of a development modulating treatment to a maize plant.In constructing and using a Relative Development Scale, the specificflower development stage used to construct the Relative DevelopmentScale may be at pollen shed for about 50 percent of a population ofmaize plants. An exemplary range on a Relative Development Scale usefulwith the method of the invention is about 0.62 to about 0.75 on aRelative Development Scale.

Determinations of the timing of tassel development may be useful foragricultural methods involving planning and/or standardizing practicesthat are plant-development specific. Examples of this include: methodsrequiring the timely application of a chemical agent, such asapplication of an herbicide, fungicide, fertilizer, and/or growthregulator across inbreds with contrasting maturities; methods requiringmonitoring, prediction, and/or adjustment of tassel development, such asmonitoring male inbreds for early tassel development, which may resultin decreased pollen shed, and providing appropriate treatment in orderto affect tassel development; methods requiring the timely applicationof a hormone and/or growth regulator to correct an imbalance and/or toproduce a desired agricultural result; and/or any methods requiringadministering a development modulating treatment to a maize plant atsaid desired tassel development stage. The invention may be used infield planning and/or research work, such as for predicting workrequirements associated with detasseling or plant development; foranticipating requirements linked to tassel development; for determininghow stress affects tassel development; and/or for use in screening forand assessing traits and/or inbreds or hybrids by imposing stress at aspecific developmental stage(s) determined by predicting the tasseldevelopment stage.

The invention provides methods useful for determining when a developmentmodulating treatment is optimally efficacious using the RelativeDevelopment Scale. As used herein, the term “development modulatingtreatment” refers to the administration of at least one physicaltreatment and/or chemical agent that affect(s) the development of aplant. Development of a plant includes, but is not limited to, flowerdevelopment, root development, leaf development, stem development,tassel development, reproductive development, gamete development, pollendevelopment, seed development, and/or the development of any other part.The modulating treatment may cause development to be terminated,retarded, prevented, delayed, or enhanced. A development modulatingtreatment may be a physical treatment, such as detasseling, flaming (useof a flame torch to singe the tops of a male plant as a means ofdelaying maturation), and/or abrading, rubbing, scraping, scratching,cutting, piercing, sonicating, detaching, breaking, removing, crushing,pruning, and/or covering any plant part. A development modulatingtreatment may be a chemical agent such as natural compounds or syntheticcompounds. Chemical agents that may be useful as a developmentmodulating treatment include plant growth regulators, plant growthregulator inhibitors, plant hormones, plant hormone inhibitors, plantgrowth stimulators, plant growth retardants, fungicides, insecticides,herbicides, auxins, antiauxins, cytokinins, defoliants, ethyleneinhibitors, ethylene releasers, gibberellins, morphactins, andgametocides. An exemplary physical treatment for use in the methods ofthe invention is detasseling and/or flaming. An exemplary chemical agentfor use in the methods of the invention is the herbicide glyphosate. Thedevelopment modulating treatment may be applied to a maize plant at anystage, for example when the tassel development stage corresponds to therange of 0.62 and 0.75 on the Relative Development Scale, which includesthe tassel development stage of T5. The ability to identify theoptimally efficacious period for the application of a tassel developmentmodulating treatment using the Relative Development Scale provided isone aspect of the invention. Many tassel development modulatingtreatments, including chemical agents are capable of preventingdevelopment of pollen or preventing pollen shed, are known in the artand would be useful in practicing the methods of the invention. Theinvention may be used for producing hybrid seed using the methods of theinvention. The invention provides methods whereby a first parental maizeplant is crossed with a second parental maize plant, wherein pollenproduction of the first parental maize plant is inhibited by applicationof a development modulating treatment. The methods of the invention maybe used to determine a development stage and/or time for application ofthe development modulating treatment to be optimally efficacious. Theinvention may be of particular use in the methods provided in U.S. Pat.No. 6,762,344 and U.S. Patent Application Publication No. 2009/0165166.In one embodiment, the invention is a hybrid seed produced employing themethods of the invention, including plants and plant parts grown fromthe hybrid seed and commodity products produced therefrom.

The invention provides a method of using transgenic maize event MON87427 plants for hybrid seed production, wherein glyphosate is used as atassel development modulating treatment in MON 87427 plants to preventpollen formation. This is predicated on the ability to prevent pollenshed in female parental lines comprising MON 87427 by timely applicationof glyphosate, thus preventing self-pollination from occurring. If theglyphosate is applied too early relative to tassel development, the malereproductive bodies may not be developed enough for the treatment to beentirely efficacious. If applied too late relative to tasseldevelopment, anther extrusion may already be underway, and theglyphosate treatment may not be able to prevent pollen development.Thus, timing of the glyphosate application relative to tasseldevelopment is important in ensuring maximum efficacy and thereforemaximum purity of the hybrid seeds produced. In one embodiment, theoptimal timing of glyphosate application for this method may beidentified by determining the timing of tassel development of MON 87427plants using a Relative Development Scale and selecting a range on theRelative Development Scale that indicates maturation to a desired tasseldevelopment stage. This determination of the timing of tasseldevelopment may then be used to identify the timing for administrationof glyphosate as a development modulating treatment to a MON 87427plant, thereby preventing self-fertilization and enhancing hybrid seedproduction. The methods of the invention reveal that tassel developmentin the range of 0.62 and 0.75 on the Relative Development Scale, or T5,is the optimal range for the application of glyphosate to MON 87427plants to prevent pollen formation. Therefore, in the Roundup®Hybridization System, for any given parental female line of any givenmaturity group, the optimal time will be predicted by multiplying theGDUs required to achieve P50 for that parental female line by any valuewithin that range. When the result of that calculation is equivalent tothe GDU's of that growing season, the optimal glyphosate applicationtime has been realized. Relative Development Scales may be producedusing other pollen shed benchmarks, and other T-Scale benchmarks thatwill be similarly useful without departing from the scope of theinvention.

Yet another aspect of the invention provides a method that is useful indetermining the tassel development stage in which reproductive crossingis optimal. Similar to the way developmental differences acrossgenotypes precludes reliable prediction of optimal modulating treatmentsbased on V-Stages or GDU's alone, timing of cross-pollination may alsobe benefited by the Relative Development Scale. A simple study using theRelative Development Scale may reveal cross-pollination is optimalwithin a certain range on that scale. Again, by knowing the GDU'srequired for P50 of a given genotype, it will be possible to pinpointwhen that range is reached without relying on unreliable vegetativebenchmarks, or performing time consuming physical assessments of tasseldevelopment. Descriptions of breeding methods that are commonly used fordifferent traits and crops can be found in one of several referencebooks (Allard, “Principles of Plant Breeding,” John Wiley & Sons, NY, U.of CA, Davis, Calif., 50-98, 1960; Simmonds, “Principles of cropimprovement,” Longman, Inc., NY, 369-399, 1979; Sneep and Hendriksen,“Plant breeding perspectives,” Wageningen (ed), Center for AgriculturalPublishing and Documentation, 1979; Fehr, In: Soybeans: Improvement,Production and Uses, 2nd Edition, Manograph., 16:249, 1987; Fehr,“Principles of variety development,” Theory and Technique, (Vol 1) andCrop Species Soybean (Vol 2), Iowa State Univ., Macmillian Pub. Co., NY,360-376, 1987).

In practicing the methods of the invention, one or both of the maizeparent plants may comprise one or more desirable trait(s) of agronomicinterest. For example, a MON 87427 parent maize plant may be used inhybrid seed production for breeding with a second parent maize plant,which comprises at least one gene and/or trait of agronomic interest. Inthis embodiment, the Relative Development Scale may be used to monitorand/or determine the reproductive development stage of the MON 87427parent maize plant in order to accurately time the treatment of the MON87427 parent maize plant with glyphosate prior to pollen formation,thereby preventing self-fertilization. The event MON 87427 plants wouldthen be pollinated by the second parent maize plant and hybrid maizeseed would be harvested from the event MON 87427 plants, wherein theseed harvested from the treated MON 87427 plants has a higher yield ofhybrid maize seed (i.e., higher percentage of hybrid seed harvested orhigher hybrid seed purity) than maize seed harvested from untreated oran inaccurately-timed treatment of event MON 87427 plants or from othermaize plant(s) under the same conditions.

Traits and genes of agronomic interest are well known in the art andinclude, but are not limited to, for example those for herbicideresistance, male sterility, increased yield, insect control, fungaldisease resistance, virus resistance, nematode resistance, bacterialdisease resistance, plant growth and development, starch production,modified and/or high oil(s) production, modified fatty acid(s) content,high protein production, fruit ripening, enhanced animal and/or humannutrition, biopolymers, environmental stress, pharmaceutical peptidesand secretable peptides, improved processing traits, improveddigestibility, low raffinose, industrial enzyme production, improvedflavor, nitrogen fixation, hybrid seed production, fiber production,and/or biofuel production. Examples of plants having one or moredesirable traits are those registered with the United States Departmentof Agriculture Animal and Plant Health Inspection Service (APHIS) forherbicide tolerance (e.g., maize events MON 88017, NK603, DAS-68416-4,HCEM485, DP-098140-6, DP-356043-5, MIR604, 59122, TC-6275, Line 1507,MON 802, T14, and/or T25), insect control (e.g. maize events MON 863,MON 809, MON 810, MON 89034, MON 88017, MON 802, MIR-162, TC-6275,DBT418, B16, TC-1507, DAS 59122-7, MIR604, and/or MON 80100), and/orother desirable traits (e.g. maize events LY038, MON 87460, and/or 3272)(a complete listing and description of such traits is available at theUnited States government's websitehttp://www.aphis.usda.gov/brs/not_reg.html).

In practicing the methods of the invention, an inbred, variety, orhybrid, or any other genotype may be used. For example, an elite inbredis a maize plant line that has resulted from breeding and selection forsuperior agronomic performance. The genotypes can be transformed and/orused in breeding methods to comprise a gene of agronomic interest suchas glyphosate tolerance and events may be selected for suitability as afemale or male parent in a hybrid seed production system.

Maize elite genotypes for use in practicing the invention, include, butare not limited to, CI9805 (U.S. Patent Publication No. 20030093826);LH321 (U.S. Patent Publication No. 20030106086); HOI002 (U.S. PatentPublication No. 20030154524); HOI001 (U.S. Patent Publication No.20030172416); 5750 (U.S. Patent Publication No. 20030177541); G0502(U.S. Patent Publication No. 20030177543); G1102 (U.S. PatentPublication No. 20030177544); HX879 (U.S. Patent Publication No.20040068771); 6803 (U.S. Patent Publication No. 20040088767); 5020 (U.S.Patent Publication No. 20040088768); G3001 (U.S. Patent Publication No.20040098768); LH268 (U.S. Patent Publication No. 20040111770); LH311(U.S. Patent Publication No. 20040111771); LH306 (U.S. PatentPublication No. 20040111772); LH351 (U.S. Patent Publication No.20040111773); LHE323 (U.S. Patent Publication No. 20040111774); 402A(U.S. Patent Publication No. 20040123352); 366C (U.S. Patent PublicationNo. 20040139491); NP2315 (U.S. Patent Publication No. 20040143866);PH0GC (U.S. Patent Publication No. 20040194170); SE8505 (U.S. PatentPublication No. 20050015834); D201 (U.S. Patent Publication No.20050028236); BE1146BMR (U.S. Patent Publication No. 20050076402); PHCAM(U.S. Patent Publication No. 20050114944); PHCK5 (U.S. PatentPublication No. 20050114945); PHC77 (U.S. Patent Publication No.20050114951); PHCND (U.S. Patent Publication No. 20050114952); PHCMV(U.S. Patent Publication No. 20050114953); PHB00 (U.S. PatentPublication No. 20050114955); PHCER (U.S. Patent Publication No.20050114956); PHCJP (U.S. Patent Publication No. 20050120437); PHADA(U.S. Patent Publication No. 20050120439); PHB8V (U.S. PatentPublication No. 20050120443); 6XN442 (U.S. Patent Publication No.20050125864); 4XP811 (U.S. Patent Publication No. 20050125865); PHCCW(U.S. Patent Publication No. 20050125866); MN7224 (U.S. PatentPublication No. 20050132433); BE9514 (U.S. Patent Publication No.20050132449); PHCAS (U.S. Patent Publication No. 20050138697); PHCPR(U.S. Patent Publication No. 20050144687); PHAR1 (U.S. PatentPublication No. 20050144688); PHACV (U.S. Patent Publication No.20050144689); PHEHG (U.S. Patent Publication No. 20050144690); NP2391(U.S. Patent Publication No. 20050160487); PH8WD (U.S. PatentPublication No. 20050172367); D501 (U.S. Patent Publication No.20050177894); D601 (U.S. Patent Publication No. 20050177896); D603 (U.S.Patent Publication No. 20050177904); PHCEG (U.S. Patent Publication No.20050223443); W16090 (U.S. Patent Publication No. 20050273876); M10138(U.S. Patent Publication No. 20050273877); N61060 (U.S. PatentPublication No. 20050273878); NP2460 (U.S. Patent Publication No.20060048243); BS112 (U.S. Patent Publication No. 20060070146); PHDWA(U.S. Patent Publication No. 20060107393); PH8JV (U.S. PatentPublication No. 20060107394); PHEWW (U.S. Patent Publication No.20060107398); PHEDR (U.S. Patent Publication No. 20060107399); PHE67(U.S. Patent Publication No. 20060107400); PHE72 (U.S. PatentPublication No. 20060107408); PHF1J (U.S. Patent Publication No.20060107410); PHE35 (U.S. Patent Publication No. 20060107412); PHEHR(U.S. Patent Publication No. 20060107415); PHDPP (U.S. PatentPublication No. 20060107416); PHEHC (U.S. Patent Publication No.20060107418); PHANF (U.S. Patent Publication No. 20060107419); PHC78(U.S. Patent Publication No. 20060107420); PH8T0 (U.S. PatentPublication No. 20060107421); PHDRW (U.S. Patent Publication No.20060107422); PHEGV (U.S. Patent Publication No. 20060107423); PHEBA(U.S. Patent Publication No. 20060107426); PHENE (U.S. PatentPublication No. 20060112463); PHEJW (U.S. Patent Publication No.20060112464); PHAPT (U.S. Patent Publication No. 20060112465); PHCND(U.S. Patent Publication No. 20060130188); PHCEG (U.S. PatentPublication No. 20060130189); PHADA (U.S. Patent Publication No.20060130190); PHEED (U.S. Patent Publication No. 20060143744); PHHB(U.S. Pat. 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No. 6,420,634); G1500 (U.S. Pat.No. 6,420,635); FR3311 (U.S. Pat. No. 6,420,636); U.S. Pat. No.1,389,972 (U.S. Pat. No. 6,420,637); PH77C (U.S. Pat. No. 6,423,888);IT201 (U.S. Pat. No. 6,426,451); G3000 (U.S. Pat. No. 6,426,453);94INK1B (U.S. Pat. No. 6,429,363); PH3HH (U.S. Pat. No. 6,433,259);6TR512 (U.S. Pat. No. 6,433,260); 89AHD12 (U.S. Pat. No. 6,433,261);U.S. Pat. No. 1,889,291 (U.S. Pat. No. 6,433,262); 2070BT (U.S. Pat. No.6,437,223); 3323 (U.S. Pat. No. 6,437,224); G1900 (U.S. Pat. No.6,441,279); 16IUL6 (U.S. Pat. No. 6,441,280); 7RN401 (U.S. Pat. No.6,444,881); UBB3 (U.S. Pat. No. 6,444,882); 6077 (U.S. Pat. No.6,444,883); U.S. Pat. No. 1,014,738 (U.S. Pat. No. 6,444,884); TDC1(U.S. Pat. No. 6,452,074); GF6151 (U.S. Pat. No. 6,452,075); 7180 (U.S.Pat. No. 6,452,076); WQDS7 (U.S. Pat. No. 6,455,764); X532Y (U.S. Pat.No. 6,459,021); U.S. Pat. No. 1,465,837 (U.S. Pat. No. 6,459,022); 1784S(U.S. Pat. No. 6,469,232); LH176Bt810 (U.S. Pat. No. 6,469,233); 6RC172(U.S. Pat. No. 6,469,234); 3327 (U.S. Pat. No. 6,469,235); 7SH382 (U.S.Pat. No. 6,476,298); U.S. Pat. No. 1,181,664 (U.S. Pat. No. 6,476,299);NP2010 (U.S. Pat. No. 6,483,014); FR3361 (U.S. Pat. No. 6,483,015);1778S (U.S. Pat. No. 6,486,386); U.S. Pat. No. 1,362,697 (U.S. Pat. No.6,492,581); RPK7250 (U.S. Pat. No. 6,506,964); and 6RT321 (U.S. Pat. No.6,911,588).

As used herein, the term “comprising” means “including but not limitedto”.

The following examples are included to demonstrate examples of certainpreferred embodiments of the invention. It should be appreciated bythose of skill in the art that the techniques disclosed in the examplesthat follow represent approaches the inventors have found function wellin the practice of the invention, and thus can be considered toconstitute examples of preferred modes for its practice. However, thoseof skill in the art should, in light of the present disclosure,appreciate that many changes can be made in the specific embodimentsthat are disclosed and still obtain a like or similar result withoutdeparting from the spirit and scope of the invention.

EXAMPLES Example 1: Transformation of Maize and MON 87427 EventSelection

The maize plant MON 87427 was produced by Agrobacterium-mediatedtransformation of maize. Maize cells were transformed and regeneratedinto intact maize plants and individual plants were selected from thepopulation of plants that showed integrity of the transgene expressioncassette and resistance to glyphosate. From this population, maize plantevent MON 87427 was selected and characterized.

The transgenic glyphosate tolerant maize plant MON 87427 was developedthrough Agrobacterium-mediated transformation of maize immature embryosutilizing transformation vector pMON58401. The transgene insert andexpression cassette of MON 87427 comprises the promoter and leader fromthe cauliflower mosaic virus (CaMV) 35S containing a duplicated enhancerregion (P-e35S); operably linked to a DNA leader derived from the firstintron from the maize heat shock protein 70 gene (I-HSP70); operablylinked to a DNA molecule encoding an N-terminal chloroplast transitpeptide from the shkG gene from Arabidopsis thaliana EPSPS (Ts-CTP2);operably linked to a DNA molecule derived from the aroA gene from theAgrobacterium sp. strain CP4 and encoding the CP4 EPSPS protein;operably linked to a 3′ UTR DNA molecule derived from the nopalinesynthase (T-NOS) gene from Agrobacterium tumefaciens.

Maize cells can be transformed by a variety of methods. For example, thefollowing method can be used to produce a transgenic maize plantcomprising the plant expression cassette of the invention. Liquidcultures of Agrobacterium tumefaciens containing the plant expressioncassette are initiated from glycerol stocks or from a freshly streakedplate and grown overnight at 26° C.-28° C. with shaking (approximately150 revolutions per minute, rpm) to mid-log growth phase in liquid LBmedium, pH 7.0, containing 50 mg/l (milligram per liter) kanamycin, andeither 50 mg/l streptomycin or 50 mg/l spectinomycin, and 25 mg/lchloramphenicol with 200 μM acetosyringone (AS). The Agrobacterium cellsare resuspended in the inoculation medium (liquid CM4C, as described inTable 8 of U.S. Pat. No. 6,573,361) and the cell density is adjustedsuch that the resuspended cells have an optical density of 1 whenmeasured in a spectrophotometer at a wavelength of 660 nm (i.e., OD₆₆₀).Freshly isolated immature maize embryos are inoculated withAgrobacterium and co-cultured 2-3 days in the dark at 23° C. The embryosare then transferred to delay media (N6 1-100-12; as described in Table1 of U.S. Pat. No. 5,424,412) supplemented with 500 mg/l Carbenicillin(Sigma-Aldrich, St Louis, Mo.) and 20 μM AgNO₃) and incubated at 28° C.for 4 to 5 days. All subsequent cultures are kept at this temperature.

The embryos are transferred to the first selection medium (N61-O-12, asdescribed in Table 1 of U.S. Pat. No. 5,424,412), supplemented with 500mg/l Carbenicillin and 0.5 mM glyphosate. Two weeks later, survivingtissues are transferred to the second selection medium (N61-0-12)supplemented with 500 mg/l Carbenicillin and 1.0 mM glyphosate.Surviving callus is subcultured every 2 weeks for about 3 subcultures on1.0 mM glyphosate. When glyphosate tolerant tissues are identified, thetissue is bulked up for regeneration. For regeneration, callus tissuesare transferred to the regeneration medium (MSOD, as described in Table1 of U.S. Pat. No. 5,424,412) supplemented with 0.1 μM abscisic acid(ABA; Sigma-Aldrich, St Louis, Mo.) and incubated for two weeks. Theregenerating calli are transferred to a high sucrose medium andincubated for two weeks. The plantlets are transferred to MSOD media(without ABA) in a culture vessel and incubated for two weeks. Rootedplants with normal phenotypic characteristics are selected andtransferred to soil for growth and further assessment. The R0 plantsgenerated through the above transformation are transferred to soil forgrowth and then selfed to produce R1 seed. Plants are selected by acombination of analytical techniques, including TaqMan, PCR analysis,and herbicide spray.

The MON 87427 event was selected from 45 individual transgenic eventsbased on multi-year analyses demonstrating the superior phenotypic andmolecular characteristics of the event and its desirable haplotypeassociation (Cr 9, 60 cM). The selection process for event MON 87427began with transformed maize plants representing 45 R0 events. Thesewere sprayed with glyphosate (64 oz/acre at V7) and then evaluated forvegetative tolerance and post-glyphosate spray sterility. Of the initial45 events, 35 R0 events exhibited vegetative glyphosate tolerance andwere male sterile when sprayed with the tested rate and timing ofglyphosate. Plants of these 35 R0 events were then advanced for furthercharacterization by molecular analysis. Using Taqman® PCR analysis andSouthern blot analysis, the 35 events were molecularly characterized. Ofthe 35 events analyzed, 29 events were selected for further advancementand field testing. The 29 R1 events were then analyzed in field trialsfor field efficacy and yield. In addition to this, additional molecularcharacterization, including genomic and protein expressioncharacterization, was done. Data were analyzed and from thecomprehensive R1 plant analysis and the field trial results, three leadevents were selected and advanced to R2 field testing. Subsequentanalysis and testing of these 3 lead events led to the selection ofevent MON 87427.

Additional field screening included treating event MON 87427 withglyphosate at 32 oz/acre (Roundup Ultra®, Monsanto Co., St. Louis, Mo.)at vegetative 4 growth stage (V4) and vegetative 10 growth stage (V10).Positive and negative plant counts were taken after the V4 spray.Additionally, treated plants were scored for chlorosis and malformationat 10-14 days after treatment (DAT) following both the V4 and V10 spray.Tassel sterility ratings at flowering were also scored for the plantssprayed at V4 and V10.

Example 2: Characterization of MON 87427 DNA Sequences

The DNA inserted into the genome of maize plant MON 87427 and theflanking genomic sequence was characterized by detailed molecularanalyses. These analyses included: sequencing the transgene insert DNAand the genomic DNA flanking the transgene insert, determining thetransgene insert number (number of integration sites within the maizegenome), determining the copy number (number of copies of transgene DNAwithin one locus), analyzing the integrity of the inserted genecassette, analyzing the genomic DNA flanking the insert and theassociation of the insertion with haplotype regions of the maize genome.

Sequences flanking the transgene DNA insertion in MON 87427 weredetermined using PCR techniques. Plant genomic DNA was isolated from thetransgenic line from tissue grown under standard greenhouse conditions.Plant tissue was combined with liquid nitrogen and ground to a finepowder using a mortar and pestle. DNA was extracted using a Nucleon™PhytoPure™ Genomic DNA extraction kit (RPN8511, Amersham, Piscataway,N.J.) according to the manufacturer's protocol. After the finalprecipitation step, DNA was resuspended in 0.5 ml of TE (10 mM Tris-HClpH 8.0, 1 mM EDTA). This method can be modified by one skilled in theart to extract DNA from any tissue of maize, including, but not limitedto, seed. An aliquot of DNA was digested with restriction endonucleasesselected based upon restriction analysis of the transgene DNA. Afterself-ligation of restriction fragments, PCR was performed using primersdesigned from the transgene DNA sequence that would amplify sequencesextending away from the 5′ and 3′ ends of the transgene DNA. PCRproducts were separated by agarose gel electrophoresis and purifiedusing a QIAGEN gel purification kit (Qiagen, Valencia, Calif.). Thesubsequent DNA products were sequenced directly using standard DNAsequencing protocols. The 5′ flanking sequence which extends into theright border (RB) sequence of the expression cassette transgene DNA ispresented as SEQ ID NO: 7. The 3′ flanking sequence which extends intothe left border (LB) sequence of the expression cassette transgene DNAis presented as SEQ ID NO: 8. The sequence fully integrated into themaize genomic DNA and containing the expression cassette DNA ispresented as SEQ ID NO: 9.

Isolated DNA molecule sequences were compared to the transgene DNAsequence to identify the flanking sequence and the co-isolated transgeneDNA fragment. Confirmation of the presence of the expression cassettewas achieved by PCR with primers designed based upon the deducedflanking sequence data and the known transgene DNA sequence. The wildtype sequence corresponding to the same region in which the transgeneDNA was integrated in the transformed line was isolated using primersdesigned from the flanking sequences in MON 87427. The PCR reactionswere performed using the Elongase® amplification system (Invitrogen,Carlsbad, Calif.). The flanking DNA sequences in MON 87427 and the wildtype sequence LH198 were analyzed against multiple nucleotide andprotein databases. This information was used to examine the relationshipof the transgene to the plant genome and to look for the insertion siteintegrity.

Example 3: Methods Useful for Identification of MON87427 DNA in a Sample

This example describes methods useful to identify event MON 87427 DNA ina sample. The event flanking sequence(s), wild-type maize genomicsequence, and/or the transgene sequence may be used to design primersand probes for use in such methods. Internal control primer(s) andprobe(s) may or may not be included in an assay.

Endpoint TAQMAN® thermal amplification methods to identify event MON87427 (event-specific assay) and/or the CP4-EPSPS synthetic gene (a.k.a.CP4-Zm) (transgene-specific assay) of event MON 87427 in a sample aredescribed. The event flanking sequence(s), wild-type maize genomicsequence, and the transgene sequence were used to design primers andprobes for use in these assays (Table 1). The DNA primers used in theevent-specific assay are primers SQ12763 (SEQ ID NO: 17) and SQ12886(SEQ ID NO: 18) with 6-FAM™ labeled probe PB4352 (SEQ ID NO: 19). TheDNA primers used in the transgene-specific assay are primers SQ20052(SEQ ID NO: 11) and SQ20053 (SEQ ID NO: 12) with 6-FAM™ labeled probePB10016 (SEQ ID NO: 13). 6-FAM™ is a fluorescent dye product of AppliedBiosystems (Foster City, Calif.) attached to the DNA probe. The controlsfor this analysis should include a positive control from maizecontaining event MON 87427 DNA, a negative control from nontransgenicmaize, and a negative control that contains no template DNA.

Additionally, an optional control for the PCR reaction may includeInternal Control Primers and an Internal Control Probe, specific to asingle copy gene in the maize genome. One of skill in the art will knowhow to design primers specific to a single copy gene in the maizegenome. The DNA primers used in the transgene-specific assay as internalcontrols are primers SQ1241 (SEQ ID NO: 14) and SQ1242 (SEQ ID NO: 15)with VIC TAMRA labeled probe PB0084 (SEQ ID NO: 16). For thetransgene-specific assay internal control primers and probe may be usedwith optional steps 5-6 below. For the event-specific assay no internalcontrol is used.

TABLE 1  Primers and Probes SEQ CP4Zm Primer-Probes ID Description NameNO Sequence Transgene Primer-1 SQ20052 11 GGCAACCGCTCGCAAATTransgene Primer-2 SQ20053 12 ATCGCCCGGAATCCTGA Transgene 6-FAM^(TM)PB10016 13 6FAM-TTCCGGCCTTTCGGGAA Probe Internal Control SQ1241 14GCCTGCCGCAGACCAA Internal Control SQ1242 15 CAATGCAGAGCTCAGCTTCATCInternal Control  PB0084 16 VIC-TCCAGTACGTGCAGTCCC VIC ProbeTCCTCCCT-TAMRA MON 87427  Primer-Probes Description Name SequenceEvent Primer-1 SQ12763 17 CGGAAACGGTCGGGTCA Event Primer-2 SQ12886 18CTCCATATTGACCATCATACTC ATTGC Event 6-FAM^(Tm) Probe PB4352 196FAM-AATGTAGAAAATCGGGA CAAT-MGBNFQ

Examples of conditions useful with Endpoint TAQMAN® methods are asfollows:

Step 1: 18 megohm water adjusted for final volume of 10 μl.

Step 2: 5.0 μl of 2× Universal Master Mix (dNTPs, enzyme, buffer) to a1× final concentration.

Step 3: 0.5 μl Event Primer-1 (SEQ ID NO: 17) and Event Primer-2 (SEQ IDNO: 18) Mix (resuspended in 18 megohm water to a concentration of 20 uMfor each primer) to 1.0 μM final concentration (for example in amicrocentrifuge tube, the following should be added to achieve 500 μl ata final concentration of 20 uM: 100 μl of Event Primer-1 (SEQ ID NO: 17)at a concentration of 100 μM; 100 μl of Event Primer-2 (SEQ ID NO: 18)at a concentration of 100 μM; 300 μl of 18 megohm water).

Step 4: 0.2 μl Event 6-FAM™ MGB Probe (SEQ ID NO: 19) (resuspended in 18megohm water to a concentration of 10 μM) to 0.2 μM final concentration.

Step 5 (Optional): 0.5 μl Internal Control Primer-1 and Internal ControlPrimer-2 Mix (resuspended in 18 megohm water to a concentration of 20 μMfor each primer) to 1.0 μM final concentration.

Step 6 (Optional): 0.2 μl Internal Control VIC™ Probe to 0.2 μM finalconcentration (resuspended in 18 megohm water to a concentration of 10μM)

Step 7: 3.0 μl Extracted DNA (template) for each sample with one each ofthe following comprising 1. Leaf Samples to be analyzed; 2. Negativecontrol (nontransgenic DNA); 3. Negative water control (no template); 4.Positive control MON 87427 DNA.

Step 8: Thermocycler Conditions as follows: One Cycle at 50° C. for 2minutes; One Cycle at 95° C. for 10 minutes; Ten Cycles of (95° C. for15 seconds then 64° C. for 1 minute with −1° C./cycle); Thirty Cycles of(95° C. for 15 seconds then 54° C. 1 minute); final cycle of 10° C.

These assays are optimized for use with either an Applied BiosystemsGeneAmp® PCR System 9700 (run at maximum speed) or MJ Research DNAEngine PTC-225 thermal cycler. Other methods and apparatus known tothose skilled in the art that produce amplicons that identify the eventMON 87427 DNA is within the skill of the art.

SEQ ID NO: 11 and SEQ ID NO: 12 or SEQ ID NO: 17 and SEQ ID NO: 18, areeach an example of a pair of DNA molecules (a primer pair) consisting ofa first DNA molecule and a second DNA molecule different from the firstDNA molecule, wherein said first and second DNA molecules each comprisea nucleic acid molecule having a nucleotide sequence of sufficientlength of contiguous nucleotides of SEQ ID NO: 10 to function as DNAprimers when used together in an amplification reaction with DNA derivedfrom event MON 87427 to produce an amplicon diagnostic for MON 87427 DNAin a sample. These primers may be used in other polymerase chainreaction (PCR) based methods for detecting the event.

Example 4: Use of Event MON 87427 for Hybrid Seed Production

The following example describes how one may use the MON 87427 for maizebreeding purposes including using the methods described in U.S. PatentPublication No. 20090165166 and/or in U.S. Pat. No. 7,314,970.

In hybrid seed production, maize plants comprising MON 87427 are plantedin an area, such as an open field. Other parent maize plant(s) may ormay not be present in the same area. For weed control during seedproduction and in commercial fields, glyphosate may be applied to maizeplants comprising MON 87427 at vegetative stages as directed on Roundup®agricultural product labels, at the same rates used in Roundup Ready®maize events NK603 and MON 88017. For hybrid seed production, twoglyphosate applications beginning just prior and/or during tasseldevelopment stages (approximate maize vegetative growth stages rangingfrom V8 to V13) are applied to the MON 87427 plants to produce a malesterile phenotype through tissue-selective glyphosate tolerance. In ahybrid maize seed production system, the MON 87427 plants withglyphosate applied at tassel development timings will be male sterileand thus can be readily pollinated by other pollen donor (male) plants,resulting in viable hybrid maize seed carrying the gene fortissue-selective glyphosate tolerance. The pollen donor plants may ormay not be present in the same area. Pollination may be affected by anymeans known in the art, including by proximity placement of plants or byhand pollination. Only specifically timed glyphosate applicationsbeginning just prior to and/or during tassel development stages(approximate maize vegetative growth stages ranging from V8 to V13) willproduce a male sterile phenotype through tissue-selective glyphosatetolerance in MON 87427. Glyphosate is a systemic herbicide that isreadily translocated via the phloem in plants. Once glyphosate is in thephloem, it moves to areas of high meristematic activity, following atypical source to sink distribution. Pollen development in a maize planttakes approximately four weeks to complete. Early tassel growth stagesstart at the approximate maize vegetative growth stage V9, thereforeglyphosate applications made at approximately this stage and time allowmaximum translocation of glyphosate to the male reproductive tissues.Glyphosate applications made during early vegetative stages, consistentwith the application timing specified in the current Roundup®agricultural product label for weed control purposes, do not affectpollen production of MON 87427 because the sensitive male reproductivetissues are not actively developing at that time. Modifications can bemade to the glyphosate treatment conditions that are known by those inthe art of herbicide application and are within the scope of invention.

MON 87427 when crossed with another glyphosate tolerant maize event suchas maize event NK603 (U.S. Pat. No. 6,825,400) to produce hybrid seedshows no yield loss when compared to yield from the conventional NK603hybrid (see FIG. 2). A field of hybrid maize plants were treated withglyphosate in two successive sprays at 2.25 lb a.e./acre each for weedcontrol and no difference was observed in injury or male fertilitybetween the various event MON 87427 hybrids and the NK603 hybrid. Thisillustrates that the F1 hybrid plants from event MON 87427 crosses arefully tolerant to glyphosate when used for weed control.

Example 5: Measuring Tassel Development Stages

Tassel development stages are illustrated in FIG. 3, with approximatesize in millimeters shown between brackets. In the figure, Vg ismeristem at vegetative stage; T0 is switch from vegetative toreproductive; T1 is reproductive growing point visible (0.9 mm); T2 islateral branch primordia visible (1.8 mm); T3 is spikelet primordiavisible (4.1 mm); T4 is central axis and lateral axis elongation (12.9mm); T5 is beginning of anthers differentiation (41.0 mm); T6 isbeginning of pollen differentiation (175 mm); and T7 is anther exertionand pollen shed (285.0 mm). The tassel development stage of a givenplant was measured by examining the tassel at various stages ofmaturation. Using a scalpel and fine forceps under a dissecting scope,the tassel meristem was dissected away from the developing leaves. Themeristem was then cut at its base with the scalpel and assessedaccording to the tassel development stages (shown in FIG. 3) by lookingthrough a microscope.

Example 6: Vegetative Development Stage (V-Stage) Relative to TasselDevelopment Stage

This example demonstrates that tassel dry weight, tassel length, andtassel development stage vary significantly across genotypes whenmeasured relative to plant vegetative stages and plant vegetativegrowth.

Ten genotypes were planted: inbred lines LH198, LH287, O1DKD2, 19HGZI,17IDI6 and hybrids DKC 44-46, DKC 47-10, DKC 52-40, DKC 58-80, DKC63-81. The hybrids were selected to be representative of genetics thatwould present a different pattern of development. The study wasconducted in Farmer City (IL), Kearney (NE), and Williamsburg (IA). Theten genotypes were planted with a cone planter and thinned to a finalstand of 38,000 plants per acre. Plot length was 20 feet with 3 feetalleys by four rows to ensure enough plants for all treatments andreduce border effects. Data were collected to record both plantvegetative development and tassel development observations relative tothe tassel development stages.

Distinct differences in tassel development were observed among genotypesat identical respective vegetative stages (V-Stages). For example,tassel size differences between genotypes were evident at every V-stage.The average tassel length at V8 stage was 7 mm for LH198, 40.2 mm forLH287, and 47.8 mm for DKC44-46 (FIG. 4). This range in tassel lengthsize at the V8 stage represents up to a 7-fold difference between thegenotypes. At the V10 stage, the average tassel lengths for these threegenotypes were 70.1 mm, 148.2 mm, and 277.3 mm, respectively (FIG. 4).This resulted in a range of almost 4 fold difference between thegenotypes. Genetic variation in tassel growth relative to V-stages isalso obvious when examining tassel dry weight accumulation.

In a further study, seventy two inbreds were used to capture a broadrange of maturities. These inbreds were grouped into 6 maturity groupsto simplify the dissection process (Table 2). Non-traited inbreds werechosen to avoid the complexity of conducting tassel dissections onregulated fields. This experiment reflects data collected at fourdifferent field locations: Williamsburg (IA), Waterman (IL), Farmer city(IL) and Constantine (MI). Four-row by twenty feet long plots weregrown. Target final population was 38,000 plants per acre. Final standcounts were documented at V3 stage. The fifth and tenth leaves of threerepresentative plants per plot were marked to keep track of V-stages;all leaves were counted including the coleoptiles. Three representativeplants from each group were sampled at 60, 70, and 80% of average growthdevelopment units to flowering (defined as when approximately half ofthe tassels in that group were shedding pollen, represented as P50) andtassel dissections were performed as previously described.

TABLE 2 Maturity Group Group 1 Group 2 Group 3 Group 4 Group 5 Group 6Average GDU 1200 1280 1330 1370 1420 1460 to P50% First 720 768 798 822852 876 dissection Second 840 896 931 959 994 1022 dissection Third 9601024 1064 1096 1136 1168 dissection

Plants were chosen from the middle rows to avoid border plants. At themoment of the first sampling, plants at a consistent development stagewere marked to be used for the second and third sampling. The dates ofeach dissection along with the V-stage at sampling times weredocumented. The tassel stage of development was identified following theRelative Development Scale as described above. Plots continued to bemonitored through flowering and the date of P50 was registered.Vegetative stage varied across inbreds relative to tassel developmentstages. For example, V-stage at T5 (beginning of anther differentiation)ranged by more than 6.5 leaves across inbreds from 7 to 14 leaves. Thiswas approximately 64% of the overall average of 10.3 leaves to reachthat stage.

Example 7: Average GDU Relative to Tassel and Flower Development Stages

This example demonstrates that the average GDUs required to achieve agiven tassel development stage or flower stage can vary significantlyacross genotypes and that GDU therefore is not a reliable predictor oftassel development. Data was taken from the field plots and inbredplants described above. Hourly temperatures from planting throughflowering were monitored using Onset weather stations and data was usedto calculate daily cumulative GDU following the traditional method(i.e., averaging daily maximum and minimum temperatures). Data fromsampled dissections indicating tassel development stage were plottedagainst GDU requirements. Inbreds that differentiate a larger number ofleaves are generally expected to have a larger GDU requirement in orderto reach a specific tassel development stage. However, the variationobserved in V-stages to T5 discussed above did not explain all thevariation observed in GDU to T5. This could suggest that thephyllochron, defined as the time between the elongation of successiveleaves, might vary between inbreds. The results of this study showedthat GDU to achieve the T5 stage ranged by more than 400 heat or growingdegree units across inbreds; about 40% of the overall average.

A strong correlation between the GDU requirements to P50 and to a giventassel development stage across inbred varieties was observed. Usingdata from the field plots and inbred plants, average GDU requirements toP50 were recorded across inbreds and compared at given tasseldevelopment stages. GDU requirements to P50 varied from 1283 to 1645units, from the shortest to the longest maturity inbred; slightly over360 GDU difference. These differences were found to correlate withdifferences in average GDU requirements to T5 stage within inbred lines(FIG. 5).

Example 8: Constructing a Relative Development Scale

This example demonstrates construction of a standardized scale formonitoring and/or predicting tassel development. This RelativeDevelopment Scale successfully standardized maize tassel developmentstages across inbreds.

Given the strong correlation between the GDU requirements to achieve P50at a given Tassel development stage across inbred varieties as describedabove, the Relative Development Scale was developed. This was calculatedby expressing tassel growth of each genotype relative to thermal time toPollen Shed as follows:

Relative Development Scale=(GDU to Tn/GDU to Px)

Data was used from the field plots and inbred plants described above.The GDU value at a given tassel development stage (GDU to Tn) wasdivided by the number of GDUs known to be required to achieve aparticular stage of pollen shed (GDU to Px), which in this case was P50for a certain genotype. This reconciled differences in tasseldevelopment stage across genotypes. For example, the GDU requirements toreach T5 were fairly consistent on the Relative Development Scale amongall inbreds and only ranged from 69 to 75% of the GDU required to P50.Variation in regression lines of GDU requirements of various genotypesrelative to Tassel development stage compared with the more consistentregression lines of those same genotypes using the standardized T-scalewere used to assess standardization of the scale, regardless of maturitygroup (FIG. 7).

Example 9: Predicting Optimal Timing for Development ModulatingTreatment

This example demonstrates use of the Relative Development Scale todetermine the optimal timing for a chemical agent spraying regimen inorder to achieve full maize tassel sterility. In this example thechemical agent was the glyphosate herbicide Roundup® used in combinationwith MON 87427 maize plants in the Roundup® Hybridization System (RHS).Optimal spraying time was correlated with actual tassel developmentstage and complete maize tassel sterility was achieved with only asingle effective dose of Roundup®.

Thirty two inbred backgrounds comprising the MON 87427 event wereselected for the study and grouped into two maturity groups. Inbredswere planted in twenty feet rows, with three feet alleys. Row spacingwas 30 inches between plants. The rows were planted such that there werefour rows of female “tester” plants followed by two rows of malepollinator plants. Transgenic events were selected for this study thathad vegetative and female-tissue tolerance to glyphosate but notmale-reproductive tissue tolerance (i.e., tissue-selective glyphosatetolerance). The male pollinators were also male tolerant to glyphosate,while the female recipient plants were male-sensitive when treated withglyphosate. Spraying treatments were blocked and sub-grouped in twobased on the inbred maturity. Immediately before each spray, threerepresentative plants from each plot were selected and dissected in thefield. Tassel length, tassel development stage, date, and GDU at spraywere recorded. Spray treatments (SS1) of Roundup PowerMAX™ with a watervolume of 15 gallons/acre were applied once to each treatment'srespective maturity group using a high clearance sprayer. SterilitySpray treatments were applied in a range from 50% through 80% of GDUsrequired to achieve P50 (averaged within inbred maturity group) as shownas shown in Table 3, where WC=“Weed Control”; Trt 1 SS1=50% GDU to P50;Trt 2 SS1=57.5% GDU to P50; Trt 3 SS1=65% GDU to P50; Trt 4 SS1=72.5%GDU to P50; Trt 5 SS1=80% GDU to P50.

TABLE 3 WC sprays = 22 oz/acre (0.75#ae/ac) SS1 sprays = 33 oz/acre(1.25#ae/ac) Fahrenheit GDU (FGDU) V3 650 750 850 950 1050 1150 Trt 1Maturity 1 WC SS1 Maturity 2 WC SS1 Trt 2 Maturity 1 WC SS1 Maturity 2WC SS1 Trt 3 Maturity 1 WC SS1 Maturity 2 WC SS1 Trt 4 Maturity 1 WC SS1Maturity 2 WC SS1 Trt 5 Maturity 1 WC SS1 Maturity 2 WC SS1

Following all spray treatments, tassel sterility/fertility assessmentswere conducted by evaluating anther extrusion and pollen shed relativeto silk emergence. These evaluations were performed when each plot wasat specific developmental stages: 10% of plants in entry with silk(S10); 50% of plants in entry with silk (S50); 90% of plants in entrywith silk (S90); 3 days after S90 date (S90+3); and 6 days after S90date (S90+6). Plants were observed for anther extrusion (AE) andsterility was measured using an Anther Extrusion Risk index (AE Risk)which is a weighted average combining the percentage of plants in theplot showing anther extrusion with the intensity of the phenomena. Forexample, Light Partial (LP) is a tassel with 10 or fewer anthersextruding. Medium Partial (MP) is a tassel with >11 anthers up to 25%anthers extruding. Heavy Partial (HP) is a tassel which has >25% ofanthers extruding. As shown in FIG. 6, the Relative Development Scalereveals an optimal window of chemical agent efficacy for producing maizetassel sterility between 0.62 and 0.75 in which AE Risk is minimizedacross inbreds and maturity groups. This study confirms theeffectiveness of the Relative Development Scale as a tool to providespraying recommendations for implementation of a Roundup® hybridizationsystem across inbreds. This would be of particular use with MON 87427.

Example 10: Method of Hybrid Seed Production with Improved Seed Purity

Methods of hybrid seed production and the resulting seed purity weremeasured using twenty-four pilot production blocks at sites in Kearney,Nebr.; Williamsburg, Iowa; Waterman, Ill.; Farmer City, Ill.; andConstantine, Mich. Four MON 87427 blocks and two cytoplasmic tasselsterility (CMS) blocks were planted at each location. MON 87427 blocksconsisted of O1DKD2MON87427-MON89034 female×80IDM2MON88017 male, and CMSblocks consisted of O1DKD2NK603B-CMS female×80IDM2MON88017 male.

The planting pattern was a 4:1 female to male ratio on 30 inch rows.Each experimental block was ten by 100 to 150 feet long panels in size.Blocks were surrounded by 30 feet (12 rows) of male on the sides as wellas on the front and back. Blocks were at least 200 feet away from otherpotential pollen sources and isolated from each other by 45 feet. Thestudy was planted at a population of 40,000 and 38,000 for irrigated andnon-irrigated land, respectively. Both the female and male rows wereplanted at the same time. The male rows were flamed at V3 growth stageto achieve stunting growth and delay pollen shed on the male plants byalternatively flaming 20 feet row length sections. Propane flamers weremounted behind a tractor and positioned over the male rows andalternated between flaming and non-flaming roughly every 20 feet.Insecticide was used at planting to minimize variability due to insectpressure. Male rows were destroyed following pollination. All blockswere sprayed with 0.75 lb a.e./acre of Roundup PowerMax™ around V3 forweed control purposes. In addition, the MON 87427 blocks were sprayedwith two sprays at 0.75 lb a.e./acre of Roundup PowerMax™ applied at 825and 975 growing degree units (GDU) from planting. The spray volume washeld constant at 15 gallons per acre (GPA).

Tassel sterility was assessed by monitoring plants every other day fromtassel emergence through 6 days after the end of silking (P90+6 days).If breakage (pollen shed) occurred, individual plants were furthercategorized as low pollen (LP; less than 10 anthers exposed), mediumpollen (MP; 11 anthers up to 25% tassel surface area with anthersextruding), or high pollen (HP; more than 25% of tassel surface areawith anthers extruding). An Anther Extrusion risk (AE Risk) was thencalculated as:

AE Risk %=([(LPx0.25)+(MPx0.5)+(HPx1.0)]/Stand count)×100

After physiological maturity and around 30-35% kernels moisture content,a 100-ear composite sample per block was hand-harvested, following apre-determined sampling scheme to represent all panels in the block.Samples were dried and weighted to adjust final yield. A first set ofsamples was hand treated and sent for quality analysis (cold germinationand warm germination assessment using two 100-seed replications). Asecond set of samples was sent for genetic purity analysis (singlenucleotide polymorphism (SNPs) analysis) and trait purity analysis(ELISA of male specific marker). A third set of samples was used todocument seed size distribution. Pilot production blocks werecombine-harvested and yield was adjusted to 15% moisture in thedetermination of bushel per acre.

Overall, both the CMS and MON 87427 blocks exceeded maize tasselsterility and seed purity standards. Anther extrusion risk was wellbelow the desired performance standard of 0.5% even 6 days after 90% ofthe female population had exerted silks (FIG. 8). Hardly any breakagewas documented on MON 87427 blocks, but a slightly higher breakage rateat late silking stages was observed on CMS blocks. Both the female andmale maize parent plants for CMS and MON 87427 were tested for geneticpurity and results showed 100% purity. The high levels of maize tasselsterility of both the MON 87427 and the CMS parent plants produced highlevels of genetic purity and trait purity in the hybrid seed producedfrom these trials (FIG. 9). There was no statistically significantdifference for trait purity between MON 87427 and CMS, but astatistically significant difference (at p<0.05) was measured forgenetic purity between MON 87427 and CMS. The genetic purity level ofhybrid seed produced using MON 87427 and the Roundup® HybridizationSystem (RHS) was 98.7%, which was significantly higher than the geneticpurity level of hybrid seed produced using CMS (98.0%). This resulted inthe female MON 87427 parent plants producing about 0.2% less ‘selfs’ and0.5% less ‘others’ than the CMS system parent plants, demonstrating thatthat use of MON 87427 with the Roundup® Hybridization System (RHS) maybe used to improve maize seed purity in maize hybrid seed production.

A deposit of a representative sample of MON 87427 seed disclosed aboveand recited in the claims has been made under the Budapest Treaty withthe American Type Culture Collection (ATCC), 10801 University Boulevard,Manassas, Va. 20110. The ATCC accession number for this deposit isPTA-7899. The deposit will be maintained in the depository for a periodof 30 years, or 5 years after the last request, or for the effectivelife of the patent, whichever is longer, and will be replaced asnecessary during that period.

Having illustrated and described the principles of the invention, itshould be apparent to persons skilled in the art that the invention canbe modified in arrangement and detail without departing from suchprinciples. We claim all modifications that are within the spirit andscope of the appended claims.

1-38. (canceled)
 39. A recombinant DNA molecule comprising thenucleotide sequence of SEQ ID NO:1 and SEQ ID NO:2 and a sequenceencoding a 5-enolpyruvylshikimate 3-phosphate synthase (EPSPS).
 40. Therecombinant DNA molecule of claim 39, further defined as comprising thenucleotide sequence of SEQ ID NO:3 and SEQ ID NO:4.
 41. The recombinantDNA molecule of claim 39, further defined as comprising the nucleotidesequence of SEQ ID NO:5 and SEQ ID NO:6.
 42. The recombinant DNAmolecule of claim 39, further defined as comprising the nucleotidesequence of SEQ ID NO:7 and SEQ ID NO:8.
 43. A method of producing maizeseed, the method comprising crossing a maize plant comprising a nucleicacid molecule comprising a nucleotide sequence selected from the groupconsisting of SEQ ID NO:1-8, and complements thereof with itself or adifferent maize plant to produce said maize seed.
 44. A maize seedproduced by the method of claim 43, wherein the maize seed comprisessaid nucleic acid molecule.
 45. The method of claim 43, furthercomprising crossing a plant grown from said maize seed with a secondmaize plant that does not comprise said nucleic acid molecule to producea progeny plant of a subsequent generation.
 46. The method of claim 45,wherein the second maize plant comprises a transgene conferring adesirable trait.
 47. The method of claim 46, wherein the transgeneconfers herbicide tolerance, insect control, enhanced oil composition,increased water use efficiency, increased yield performance, increaseddrought resistance, increased seed quality, or improved nutritionalquality.
 48. A maize seed produced by the method of claim 47, whereinthe maize seed comprises said nucleic acid molecule.
 49. The maize seedof claim 48, wherein the maize seed is a hybrid seed.
 50. The method ofclaim 45, further comprising: (a) crossing the progeny plant of thesubsequent generation with itself to produce a progeny plant of afurther subsequent generation; (b) crossing the progeny plant of thefurther subsequent generation with itself; and (c) repeating step (b) asufficient number of times to generate an inbred plant.
 51. A maize seedproduced by the method of claim 50, wherein the maize seed comprisessaid nucleic acid molecule.
 52. The maize seed of claim 51, wherein themaize seed is an inbred seed.
 53. A method of obtaining a maize plantthat tolerates application of glyphosate herbicide comprising: (a)applying glyphosate herbicide to a plurality of maize plants, wherein atleast one of the maize plants comprises a nucleic acid moleculecomprising a nucleotide sequence selected from the group consisting ofSEQ ID NO:1-8, and complements thereof; and (b) identifying a plant thattolerates application of glyphosate herbicide.